Use of Transabdominal Ultrasound for the detection of intra-peritoneal tumor engraftment and growth in mouse xenografts of epithelial ovarian cancer.

OBJECTIVE: To evaluate intraperitoneal (IP) tumor engraftment, metastasis and growth in a pre-clinical murine epithelial ovarian cancer (EOC) model using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system (IVIS).
METHODS: Ten female C57Bl/6J mice at six weeks of age were included in this study. Five mice underwent IP injection of 5x106 ID8-luc cells (+ D- luciferin) and the remaining five mice underwent IP injection of ID8-VEGF cells. Monitoring of tumor growth and ascites was performed weekly starting at seven days post-injection until study endpoint. ID8-luc mice were monitored using both TAUS and IVIS, and ID8-VEGF mice underwent TAUS monitoring only. Individual tumor implant dimension and total tumor volume were calculated. Average luminescent intensity was calculated and reported per mouse abdomen. Tumor detection was confirmed by gross evaluation and histopathology. All data are presented as mean +/- standard deviation.
RESULTS: Overall, tumors were successfully detected in all ten mice using TAUS and IVIS, and tumor detection correlated with terminal endpoint histology/ H&E staining. For TAUS, the smallest confirmed tumor measurements were at seven days post-injection with mean long axis of 2.23mm and mean tumor volume of 4.17mm3. However, IVIS imaging was able to detect tumor growth at 14 days post-injection. Ascites formation was detected in mice at 21 days post-injection.
CONCLUSIONS: TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVIS. In addition, TAUS provides relevant information for ascites development and detection of multiple small metastatic tumor implants. TAUS provides an accurate and reliable method to detect and monitor IP EOC growth in mouse xenografts.

Introduction

Epithelial ovarian cancer (EOC) is a leading cause of gynecologic cancer related mortality in women [1]. The five-year overall survival for women with EOC is poor since the majority of patients present with advanced and metastatic disease [2]. Additionally, although patients initially respond well to treatment with surgery and chemotherapy with carboplatin and paclitaxel, the vast majority of women will recur [4-8]. Ovarian carcinomas primarily undergo peritoneal dissemination, and are often associated with malignant ascites. This pattern of spread is associated with vague symptoms which leads to delays in diagnosis [3]. There is a significant unmet need for methods to facilitate early diagnosis of EOC and advance current therapeutic options.

Pre-clinical research utilizing EOC cell lines and patient-derived xenografts shows tremendous promise in advancing the current understanding of EOC carcinogenesis and therapeutics [9-15]. In longitudinal pre-clinical studies, the ability to detect tumor engraftment and sequentially assess tumor volume utilizing non-invasive techniques is essential to assessing tumor growth and treatment response. However, despite the existence of many cell lines that closely replicate human EOC at a cellular level, difficulty monitoring intraperitoneal tumor formation, growth and metastasis remains a major limitation in the execution of preclinical EOC studies [9-15].

In-vivo monitoring of EOC cell lines can be accomplished using several well- developed techniques including RFP, GFP, luciferase and ROSA reporter systems [16, 17]. Bioluminescence in vivo imaging system (IVIS) using luciferase reporter containing cell lines has been commonly utilized to track tumor growth over time, but this technique has limitations. This imaging technique involves injection of luciferin in conjunction with tumor cells, which is invasive and can initiate an inflammatory response [18]. Additionally, this technique only provides qualitative information regarding tumor progression [17]. The primary concern for use of IVIS is the necessity to use modified cell lines which have a tendency for genetic drift and phenotypic alterations. As such, use of IVIS for monitoring of patient-derived xenografts is not feasible [19,20]. In addition, a pertinent characteristic of EOC patients is the development of ascites throughout the progression of disease and the accuracy of luciferase is diminished in the presence of abdominal ascites as a result of dilution [21-24]. These studies support that IVIS imaging technology has limitations in ones ability to perform in-vivo experiments utilizing PDX and cell lines prone to ascites development, which are important in EOC research. Therefore, development of novel imaging strategies to study EOC in animal models is needed.

In clinical practice, transabdominal ultrasound (TAUS) is frequently utilized in the evaluation of women with gynecologic diseases, including EOC [25,26]. Despite being non-invasive, cost-effective and accurate, data for use of TAUS for monitoring of EOC in pre-clinical murine models is limited [27]. The objective of this study was to evaluate intraperitoneal tumor engraftment and growth in the presence and absence of ascites in a pre-clinical murine model of EOC utilizing both TAUS and IVIS imaging.

Methods

Cell lines and lentiviral transformation of ID8 cells with luciferase vector

ID8 and ID8-VEGF syngeneic EOC cell lines were cultured in Dulbecco Modified Eagle Medium (DMEM) media containing heat inactivated 5% FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1) and 100 U/mL penicillin-streptomycin and 1% insulin/transferrin/selenium and grown under standard conditions. HEK 293T/17 (ATCC CRL-11268) cells were plated at 65% confluence in a 100 mm dish and cultured in 9 mL DMEM supplemented with heat inactivated 10% FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1)[15]. ID8 cells were subsequently transfected with luciferase containing construct pHIV-Luciferase #21375 4.5 μg (Addgene) to generate the ID8-luc cells. Briefly, 3 mL of the DMEM media was removed and ID8 cells were co-transfected with Lipofectamine 3000 (L3000015 Invitrogen) 35 μL of Plus reagent / 41 μL of Lipofectamine 3000, 3rd generation packaging vectors pRSV-REV #12253 4.3 μg, pMDG.2 #12259 4.3 μg, and pMDLg/pRRE #12251 4.3 μg (Addgene) and lentiviral vector directing expression of luciferase reporter pHIV-Luciferase #21375 4.5 μg (Addgene) in 3 mL of OptiMEM media. Following 8 hours of incubation, media of the 293T/T17 cultures was replaced and following 18 hours of incubation media containing viral particles were harvested and filtered through a 0.45 μm Durapore PVDF Membrane (Millipore SE1M003M00). Viral transfections were carried out over 72 hours ID8 parental cells and transduced cells were selected by their resistance to 2 μg/mL puromycin (MP Biomedicals 0219453910). Prior to use in this experiment, activity of luciferase promoter and tumor growth was confirmed in a pilot cohort of mice.

Ethics statement

All studies were carried out in accordance with protocols approved by the Institutional and Animal Care and Use Committee (approval # 2018–2003) or the Institutional Biosafety Committee (approval # IBC0920) of the Cleveland Clinic Lerner Research Institute Biological Resource Unit.

Mouse xenografts

Ten female C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 6 weeks of age. After two weeks of acclimation, mice underwent IP injection of 300μL of either 5x106 ID8-luc (n = 5) or ID8-VEGF (n = 5) cells. Following cell injection mice were monitored daily for three days then every 4 days for the duration of the study. Additionally, mice were monitored weekly by TAUS and IVIS imaging for tumor progression. Mice reached endpoint criteria and immediate CO2 asphyxiation followed by cervical dislocation, when any of the following conditions were observed: tumor burden >1.5cm2 by TAUS, tumor interfered with animals ability to eat or drink, 20% weight loss, palpation of tumor elicited a pain response, became unresponsive or death was imminent, exhibited respiratory difficulty or hypothermia, or any sign of outward distress such as hunched posture, ruffled fur, and reduced motility. All efforts were made to minimize suffering and distress including the use of dietary gel packs and placing the cage on a heated surface following anesthesia during recovery observation.

All mice were removed from the study at 60 days post cell injection. None of the mice died before meeting endpoint criteria or pre-determined 60-day endpoint of study.

Tumor monitoring

Ultrasonography was performed using a Vevo2100 (VisualSonics) with an abdominal imaging package and MS550D probe (40Hz)(Fig 1). TAUS surveillance was initiated seven days following IP tumor injection. TAUS was performed every seven days until study endpoint at 67 days. Mice were anesthetized using isoflurane (DRE Veterinary) and placed in the supine position. Following the removal of abdominal hair using Nair (Church & Dwight Co. Inc.), sterile ultrasound gel was applied to the abdomen. TAUS was performed using Vevo2100 (VisualSonics) using the abdominal imaging package and MS550D probe (40Hz) (S1 Fig). Throughout the duration of TAUS imaging, murine heart rate was monitored and heated platform was utilized to minimize distress. For each timepoint, when each individual mouse underwent ultrasound, the abdomen was assessed for tumor in four locations based on abdominal quadrants: 1) right upper, 2) right lower, 3) left upper, 4) left lower. In order to provide specific information about tumor size discrimination with ultrasound, the individual size of each tumor implant was reported. Each animal served as its own control for assessing longitudinal tumor growth. Tumors were noted to be absent or present at each assessment, as well as tumor location by quadrant (data not shown). Tumor dimensions (length and width) were recorded and tumor volume was calculated using the formula: (Length*(Width2))/2. Following mouse necropsy, tumors were submitted for H&E for confirmation. Study design is depicted in Fig 1.

Fig 1

Study design.

2D IVIS imaging

Bioluminescence images were taken within 48 hours of ultrasound images with IVIS Lumina (PerkinElmer) using D-luciferin as previously described [24]. Mice received an IP injection of D-luciferin (Goldbio LUCK-1G, 150mg/kg in 150mL) under inhaled isoflurane anesthesia. Images were normalized (Living Image Software) with a minimum and maximum radiance of 7.58105 and 5.39108 photons/second/cm2/steradian, respectively. All images were obtained with a 15 second exposure. Average luminescent intensity in photons per second/cm2/steradian was calculated and reported for each mouse abdomen.

3D IVIS imaging

Upon endpoint, bioluminescence and x-ray images were taken using the IVIS Spectrum system (PerkinElmer). Mice were sedated with 2% isoflurane (DRE Veterinary) inhalation in an airtight transparent anesthesia box for 5 minutes. Mice were shaved front and back and Nair was applied to remove remainder of the hair before being IP injected with D-luciferin (Goldbio LUCK-1G, 150mg/kg in 150 mL). Mice are placed in a supine position on the light-tight chamber of the CCD camera imaging unit. Sequential images were acquired at 1min intervals (60 s exposure, no time delay) for at least 30 min. The luminescence camera was set to 60 s exposure, medium binning, f/1, blocked excitation filter, and open emission filter. The photographic camera was set to auto exposure, medium binning, and f/8. Average luminescent intensity in photons per second/cm2/steradian was calculated and reported for each mouse abdomen. Identical settings were used to acquire each image and region of interest during the study. Ultrasound and IVIS imaging were performed independently by two separate investigators who were blinded to the results of the other imaging modality.

Patient Derived Xenograft Pilot Study

In order to assess the utility of TAUS in PDX models of EOC, the investigators performed TAUS in the manner described above in a small cohort of mice (n = 5). In this pilot experiment, tumor samples were obtained at the time of primary debulking surgery for high grade serous EOC or uterine serous carcinoma, processed into a single-cell suspension and injected via IP injection. TAUS was done at four time points– 3 days, 10 days, 14 days and 21 days post-IP injection.

Statistics

All data are presented as mean +/- standard deviation. Tumor volumes presented as mean+SEM and graphed over time. All statistical analysis was performed in GraphPad Prism v8.

Results

Tumor engraftment was detected in all C57Bl/6J via TAUS between 7–14 days, gross examination at necropsy and on histopathology. In addition, in mice injected with ID8-luc, tumor engraftment was noted at 14 days. In all cases, EOC tumors were detected before any clinical signs (ascites, palpable masses, lethargy). Beginning at Day 7 post-injection, TAUS was performed and a maximum of four tumor measurements were recorded per mouse in each abdominal quadrant. Six mice (60%) had one detectable tumor on TAUS at 7 days. All mice (n = 10) had at least one tumor detectable on TAUS at 14 days post-injection and 20% (n = 2) had two detectable tumors. Mean tumor dimensions and volumes for ID8-luc and ID8-VEGF are displayed in Table 1 and Table 2, respectively. The smallest tumor short and long axis measurements detected at 7 days were 1.74mm and 2.23mm, respectively. The lowest recorded tumor volume was 4.17mm. Ascites was detected as early as 21 days. Tumor volume detected via TAUS over time is displayed for both ID8 and ID8 VEGF mice in Fig 2. Fig 3 depicts weekly TAUS images of EOC tumor implant over time in ID8-luc without ascites (A) and in ID8-VEGF (B). To test whether TAUS can be used to detect PDX tumor, we injected mice (n = 5) with a PDX single cell suspension of EOC human cells. We were able to detect tumor growth for a human PDX tumor of high grade serous EOC at 10 days post injection (1.893mm in long axis). Longitudinal imaging demonstrated increased growth on days 14 (2.200mm in long axis) and 21 (3.875mm in long axis), respectively. (S2 Fig).

Table 1

Mean tumor measurements in ID8 mice with TAUS and IVIS imaging.

	Transabdominal Ultrasound Measurement				IVIS Imaging
	Mean Tumor Implant Long Axis (mm) (n = 20)	Mean Tumor Implant Short Axis (mm) (n = 20)	Mean Tumor Implant Volume (mm3) (n = 20)	Mean Total Tumor Burden per Mouse (n = 5) (mm3)		photon/second/cm^2/sr
Day 7	2.83	1.80	4.61	4.61	Day 7	2.63e6
Day 14	2.92	2.11	6.81	9.55	Day 14	1.11e6
Day 21	4.10	2.58	14.5	43.38	Day 24	4.15e6
Day 28	4.26	2.76	17.28	69.10	Day 31	4.88e6
Day 35	4.94	3.50	32.54	130.14	Day 39	7.75e6
Day 42	5.88	4.14	51.34	205.35	Day 46	1.95e7
Day 49	7.20	4.58	75.58	305.53	Day 57	2.18e7
Day 56	8.56	5.06	110.00	432.83

Table 2

Tumor measurements in ID8-VEGF mice with transabdominal ultrasound.

	Transabdominal Ultrasound Measurement
	Mean Tumor Implant Long Axis (mm) (n = 20)	Mean Tumor Implant Short Axis (mm) (n = 20)	Mean Tumor Implant Volume (mm3) (n = 20)	Mean Total Tumor Burden per Mouse (n = 5) (mm3)	Presence of Ascites
Day 7	2.23	1.98	4.39	4.39	No
Day 14	3.00	1.90	5.56	6.79	No
Day 21	3.98	2.54	13.52	35.23	Yes
Day 28	4.30	2.70	16.73	49.92	Yes
Day 35	5.36	3.70	38.59	130.28	Yes
Day 42	6.55	4.03	56.09	178.81	Yes

Fig 2

Transabdominal ultrasound allows for monitoring of tumor engraftment and growth in mice with ovarian cancer xenografts of ID8 (A) and ID8-VEGF (B).

Fig 3

Transabdominal ultrasound allows for longitudinal monitoring of intraperitoneal tumor implants in murine model of EOC.

Within 48 hours of TAUS, 2D IVIS imaging was performed. ID8 tumor detection by 2D IVIS imaging was noted at 7 days post cell injection and intraperitoneal tumor growth over time was tracked as previously reported (Fig 4A and 4B). As the PDX and VEGF cell lines do not contain a luciferase reporter system, IVIS imaging was not performed on these cohorts. At Day 14 it was noted (Table 1) that the photon/second/cm^3/sr read was decreasing and then increased at subsequent timepoints as expected. This speaks to the high variance of IVIS at early timepoints, and why having an added system of tumor progression analysis, such as TAUS, is essential. Additionally, 3D IVIS imaging including murine x-ray was performed at endpoint to determine tumor location (Fig 4C). All tumors were located in regions identified by 3D IVIS imaging at endpoint necropsy (data not shown).

Fig 4

2D IVIS imaging tracked tumor growth over time, and 3D IVIS imaging determined endpoint tumor volume in ID8 tumor bearing C57Bl/6 mice.

Prior to necropsy, the murine abdominal cavity was imaged to confirm gross tumor presence in the ID8 and ID8 VEGF cohort. Each tumor was then excised and stained using H&E to confirm EOC histology (Fig 5). Following murine necropsy, ID8 tumors were imaged and excised (Fig 5). ID8 and ID8 VEGF EOC tumor phenotype was confirmed by histology (Fig 5A and 5B respectively).

Fig 5

Upon necropsy macroscopic and histologic EOC tumors were identified and validated for both ID8 and ID8 VEGF cell lines.

Discussion

Mouse xenografts represent an important method to pursue urgently needed preclinical studies to understand pathogenesis and develop new therapies for EOC. While many orthotopic models exist that closely mirror human EOC, techniques to monitor intraperitoneal tumors in an accurate, non-invasive fashion are limited. In this study, we applied ultrasonography, to evaluate the engraftment and growth of EOC in a pre-clinical model. We demonstrated that in murine models of EOC, TAUS can be used to accurately detect and monitor the growth of EOC xenografts with tumors and ascites detected as early as 7 and 21 days post-injection, respectively. We found TAUS is more sensitive for detection of disease progression compared to bioluminescence assays where tumor detection first occurred at 14 days post-injection. Our findings are consistent with prior studies demonstrating that IVIS is able to detect tumor growth at 2 weeks after ID8-luc cell injection [21].

Currently utilized and previously described strategies for tumor monitoring in murine models of EOC fall short [9–15, 21–24]. IVIS imaging is frequently used for tumor assessment in murine models of EOC but has significant limitations. First, the cell-line must contain a luciferase reporter, which limits the ability to utilize high fidelity patient-derived tumorgraft models. Second, concerns exist regarding initiation of an inflammatory response or other phenotypic and genotypic alterations that may render the cell line less applicable to human EOC [19-21,23]. Finally, detection of ascites is compromised in IVIS models. Baert et al demonstrated that reduced sensitivity of IVIS in the presence of luciferase with a significantly decreased in the presence of ascites within an ID8-luc model [23]. As the majority of human and mouse EOC lines have a penchant for ascites development, the detection of ascites is of high importance. In the clinical setting, ascites significantly impacts patient quality of life and is a harbinger of advanced, progressive disease. Ascites is important to study in pre-clinical translational models as it can yield diagnostic and prognostic information.

In clinical practice, TAUS is frequently utilized in the evaluation of women with gynecologic diseases, including EOC [25,26]. However, prior to this study, application of ultrasonography to murine pre-clinical EOC models has been limited. Weroha et al. utilized ultrasonography to assess tumor growth in patient-derived xenografts of EOC with high correlation between ultrasound assessment and tumor measurements at necropsy [27]. In addition, TAUS has been utilized in pre-clinical models of non-gynecologic intra-abdominal cancers, including pancreatic and genitourinary malignancies [28-30]. Within a murine model of bladder cancer, Patel et al. demonstrated high correlation between tumor size with transabdominal micro-ultrasound and at necropsy and were able to detect tumors as small as 0.95 mm3 [30]. Similarly, in pre-clinical murine models of pancreatic adenocarcinoma, intra-pancreatic tumors were detected as early as three days post-injection, and tumor metastasis in addition to ascites was identified in all animals at two weeks with excellent correlation to necropsy tumor volume [29].

TAUS offers several potential advantages over currently available imaging tools for the monitoring of murine models of EOC. Primarily, we demonstrate in this study that tumor detection can be assessed as early as one week post-injection, with tumor implants detected as small as 2mm in longest dimension. Secondly, malignant ascites and innumerable tumor implants are pathognomonic of human EOC. This method allows researchers to monitor treatment response via tumor volume and ascites in parallel to patients undergoing chemotherapy where radiologic scoring systems such as RECIST criteria are used. In addition, TAUS can be utilized for EOC monitoring in cell lines that do not have RFP, GFP, luciferase or ROSA reporter systems. Thereby, this allows for in-vivo monitoring of any intra-peritoneal EOC cell line with or without ascites development, including PDX models. This is important because it allows researchers to follow tumor growth and treatment response over time with cells transplanted directly from patient tumor specimens without the need for luciferase transduction. Finally, the ability to accurately detect tumors may represent a strategy to minimize animal euthanasia, as their disease burden can be monitored in-vivo to end-point during an experiment without need for early necropsy with each animal serving as its own control. Therefore, monitoring EOC growth and response via TAUS has improved detection, higher sensitivity and increased breadth and utility over presently utilized imaging techniques.

In clinical practice, transabdominal and transvaginal US remain gold-standard for the initial assessment of gynecologic pathology, including ovarian tumors. TAUS is non-invasive and cost-effective with low risk to the patient. In this study, we demonstrate that this same imaging modality can be applied to mouse xenografts. Based on these results, we have adopted TAUS as a method to monitor tumor growth and treatment response in EOC preclinical studies in both syngeneic and PDX models with excellent success and reproducibility. One limitation of this model is the need for mouse anesthesia during TAUS. In this series, anesthesia and TAUS were well tolerated by the mice with no adverse intra-anesthesia events or mortalities related to the procedure. In our reported experience, the application and interpretation of TAUS imaging to murine models of EOC is feasible and is no more challenging than other imaging modalities utilized for pre-clinical tumor monitoring, including IVIS. Despite this, to the best of our knowledge, this study represents the first publication assessing the feasibility of TAUS for preclinical murine models of EOC in parallel with IVIS imaging.

In conclusion, TAUS shows promise in the detection of tumor growth and metastasis and response to therapies in intraperitoneal mouse xenografts of EOC. TAUS allows for detailed measurements of tumors and metastatic implants, ascites and is more sensitive than IVIS imaging.

Procedural steps for transabdominal ultrasound.

(TIF)

Click here for additional data file.

Human derived EOC PDX tumor detected at 10 days post IP injection (A).

(TIF)

Click here for additional data file.

5 Mar 2020

PONE-D-20-00914

Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer

PLOS ONE

Dear Dr. Reizes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Apr 19 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Shannon M. Hawkins, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2) In your Methods section, please give the sources of any cell lines used in your study (such as ID8 and ID8-VEGF).

3) Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

4) Please include a title for Tables 1 and 2.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled “Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer” by Chambers et al. have described the results detecting tumor development in ID8 mouse EOC cell injected mice using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system

(IVIS). The time-dependent detection limits reported are informative. However, this study suffers several major flaws.

1. In Introduction, the authors stated “The objective of this study was to evaluate intraperitoneal tumor engraftment and growth in the presence and absence of ascites in a pre-clinical murine model of EOC utilizing both TAUS and IVIS imaging.” This goal has a very limited scale and significance to ovarian cancer research. In particular, the IVIS has been used for mouse imaging for many years.

2. Two areas need to be included in this study to have enough significant impact and be suitable to the scale of a PlosOne publication: 1) include a human xenograft model(s) to test the detection limitation of human tumors in mice; and 2) include several known EOC markers in blood/ascites/or peritoneal washing to compare to TAUS sensitivity and specificity (by including a few control mice) in detecting early stage of EOC.

3. In particular, Much16, HE4, and VEGF in blood/ascites/or peritoneal washing should be measured at the same time points with TAUS and IVUS to compare their sensitivity and specificities.

4. Control mice are needed for TAUX and IVIS detection.

5. Importantly, the 10 mice used in study only sacrificed by the end to confirm the tumors. The TAUX imagines shown in Fig. 2 indicate that it is difficult to quantify tumors using TAUX. Validation of early-detected tumors and their measurements are critical for the study.

6. The detection ability related to tumor locations should be described.

7. The ID8-VEGF cells are much more aggressive as reported (PMID: 16886597). However, in this study, no difference or even less tumor burdens in the ID8-VEGF cell injected mice from Day 14 to Day 42. This needs to be clearly interpreted.

8. Why did the photon/second/cm^3/sr at Day 14 was reduced from Day 7 (Table 1) needs to be explained.

9. Tables 1-2 should have titles.

Reviewer #2: 1. IVIS 2D imaging, 15 sec exposure seems long even for the earlier time points. Did the author try different time of exposure?

2. Table 1-under IVIS imaging, the right part of the table, the intensity should be shown as cm^2 if it’s 2D imaging.

3. Tables 1 and 2- what does the n=20 mean under “Transabdominal ultrasound measurement”

4. Figure 3, image from day 7 is missing and should be included.

5. Consider including a figure illustrating the study design

6. Provide more details about the PDX and the PDX study- patient information, tumor information, number of cells injected, number of mice injected and any other time points assessed using TAUS or was only day 10. Did ref 27 also examine pre-necropsy time points?

7. Are there other IVIS imaging studies using the ID8-luc model that the authors can directly compare their results at similar time points?

8. Line 66: IVIS not IVUS

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

5 Apr 2020

Manuscript: PONE-D-20-00914

Title: Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer

Corresponding Author: Ofer Reizes PhD; First Author: Laura Chambers, DO

Response to Reviewers

Reviewer #1:

The manuscript entitled “Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer” by Chambers et al. have described the results detecting tumor development in ID8 mouse EOC cell injected mice using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system

(IVIS). The time-dependent detection limits reported are informative. However, this study suffers several major flaws.

1. In Introduction, the authors stated “The objective of this study was to evaluate intraperitoneal tumor engraftment and growth in the presence and absence of ascites in a pre-clinical murine model of EOC utilizing both TAUS and IVIS imaging.” This goal has a very limited scale and significance to ovarian cancer research. In particular, the IVIS has been used for mouse imaging for many years.

Response: While IVIS has been utilized for imaging in mouse models of EOC, this method does have limitations. As discussed in the introduction, IVIS necessitates a modified cell line with a promoter and studies have demonstrated that this may induce an inflammatory response. Specifically, for EOC research, IVIS is unable to be used in animal studies of patient-derived xenografts (PDX), which provide important pre-clinical data that cell lines cannot [19,20]. In addition, EOC tumors are prone to ascites development. Ascites in EOC significantly impacts survival outcomes and patient quality of life. In the presence of ascites, the accuracy of IVIS is significantly diminished due to dilution of substrate [21-24]. In our practice, we have encountered that IVIS imaging technology falls short in our ability to perform in-vivo murine experiments utilizing PDX and cell lines without promoters, especially in the early detection of tumors prior to chemotherapy initiation. The authors believe there is an un-met need to develop further imaging strategies to study EOC in animal models. Ultrasound is used widely in clinical practice for the diagnosis and management of gynecologic pathology, including ovarian cancer, and in translation research in other malignancies, including genitourinary malignancies. Therefore, the authors set out to investigate whether ultrasound can be similarly used in murine models of EOC. This has been further explained in the introduction.

Line Number(s): 125-128

Revised Text: “These studies support that IVIS imaging technology has limitations in the ability to perform in-vivo experiments utilizing PDX and cell lines prone to ascites development, which are important in EOC research. Therefore, development of alternative imaging strategies to study EOC in animal models is needed.”

2. Two areas need to be included in this study to have enough significant impact and be suitable to the scale of a PlosOne publication: 1) include a human xenograft model(s) to test the detection limitation of human tumors in mice; and 2) include several known EOC markers in blood/ascites/or peritoneal washing to compare to TAUS sensitivity and specificity (by including a few control mice) in detecting early stage of EOC.

Response: The authors performed a pilot study utilizing our gynecologic cancer biorepository with human PDX models of EOC and uterine serous carcinoma (n=5). In this experiment, mice underwent TAUS at four time points (3 days, 10 days, 14 days, 21 days), to assess the utility of TAUS in detection. Tumors were able to be detected as early as 10 days utilitizing a high grade serous EOC line in a patient who underwent primary debulking surgery for Stage IIIC disease. This has been described within the methods and results below.

Line Number(s): 207-213; 235-238

Revised Text: “In order to assess the utility of TAUS in PDX models of EOC, the investigators performed TAUS in the manner described above in a small cohort of mice (n=5). In this pilot experiment, tumor samples were obtained at the time of primary debulking surgery for high grade serous EOC or uterine serous carcinoma, processed into a single-cell suspension and injected via IP injection. TAUS was done at four time points – 3 days, 10 days, 14 days and 21 days post-IP injection.

“We were able to detect tumor growth for a human PDX tumor of high grade serous EOC at 10 days post injection (1.893mm in long axis). Longitudinal imaging demonstrated increased growth on days 14 (2.200mm in long axis) and 21 (3.875mm in long axis), respectively (Supplemental Figure 1).”

3. In particular, Much16, HE4, and VEGF in blood/ascites/or peritoneal washing should be measured at the same time points with TAUS and IVUS to compare their sensitivity and specificities.

Response: Assessment of MUC16, HE4 and VEGF in ascites and blood have demonstrated tremendous promise in the assessment of treatment response in both patients and animal models of EOC. Unfortunately, the role of these biomarkers is limited in the assessment of early stage EOC, especially prior to ascites and metastasis formation. In our study, ascites development was not detected until 21 days post tumor injection, compared to TAUS which detected ultrasound at 7 days. Specifically, high levels of HE4 in ascites has been linked to chemo-resistance (Liu) and may predict for optimal debulking in primary surgery (Paunovic) and overall survival (Braicu), but there is no data in the literature supporting HE4 should be used as a diagnostic biomarker of early stage ovarian cancer. The authors believe that the lack of useful biomarkers and imaging to detect early stage EOC supports how TAUS, which detects tumors as early as 7 days post-injection, may be a helpful tool in pre-clinical models. In addition, TAUS detects disease prior to the development of ascites and may be useful for study of metastasis, and response to chemotherapy in early stage disease.

Line Number(s): N/A

Revised Text: N/A

References:

1. Braicu EI, Fotopoulou C, Van Gorp T, Richter R, Chekerov R, Hall C, et al. Preoperative HE4 expression in plasma predicts surgical outcome in primary ovarian cancer patients: results from the OVCAD study. Gynecol Oncol. 2013 Feb;128(2):245-51.

2. Liu D, Kong D, Li J, Gao L, Wu D, Liu Y, et al. HE4 level in ascites may assess the ovarian cancer chemotherapeutic effect. J Ovarian Res. 2018 Jun 14;11(1):47,018-0402-3.

3. Paunovic V, Protrka Z, Ardalic D, Paunovic T. Usefulness of human epididymis protein 4 in predicting optimal cytoreductive therapy in patients with advanced ovarian cancer. J BUON. 2017 Jan-Feb;22(1):29-33

4. Control mice are needed for TAUX and IVIS detection.

Response: The authors did not use control animals in this study. Primarily, in this study design, each mouse serves as its own control throughout the study, which is parallel to clinical practice where individual tumors are followed and assessed for response. Secondly, based on previous studies validating use of transabdominal ultrasound for genitourinary and gastrointestinal tumors, control animals without tumors were not utilized as each animal was used as its own control [28-30]. In addition, the authors aimed to use the lowest number of animals to complete the study objectives per our institutional IACUC governance.

Line Number(s): 178-179

Revised Text: “Each animal served as its own control for assessing longitudinal tumor growth.”

5. Importantly, the 10 mice used in study only sacrificed by the end to confirm the tumors. The TAUX imagines shown in Fig. 2 indicate that it is difficult to quantify tumors using TAUX. Validation of early-detected tumors and their measurements are critical for the study.

Response: Previous studies have demonstrated that transabdominal ultrasound measurements are comparable to weights of tumors at necropsy. The objective of this study was to specifically correlate tumor measurements from IVIS and TAUS for preclinical models of EOC. In this study, we identified tumor growth as early as seven days post-injection, which is comparable to studies in gastrointestinal and genitourinary malignancies. In our results, IVIS was able to detect tumors at day 14 post-injection, which is similar to previously published data in ID8-luc cell lines. In our experience and with appropriate training of laboratory staff, we have found that the application and interpretation of TAUS imaging to murine models of EOC is feasible and is no more challenging than other imaging modalities utilized for pre-clinical tumor monitoring, including IVIS.

Line Number(s): 334-337

Revised Text: “. In our reported experience, the application and interpretation of TAUS imaging to murine models of EOC is feasible and is no more challenging than other imaging modalities utilized for pre-clinical tumor monitoring, including IVIS.”

6. The detection ability related to tumor locations should be described.

Response: There is no difference in ability to detect tumors based on location with TAUS.

Line Number(s): N/A

Revised Text: N/A

7. The ID8-VEGF cells are much more aggressive as reported (PMID: 16886597). However, in this study, no difference or even less tumor burdens in the ID8-VEGF cell injected mice from Day 14 to Day 42. This needs to be clearly interpreted.

Response: Although the ID8 VEGF does not exhibit enhanced rate of tumor growth, the debilitating ascites that develops in these mice speaks to the aggressive nature of the subtype of EOC. As such, ID8 VEGF mice had overall poorer health when compared to the ID8 cohort and were removed from the study at an earlier timepoint.

Line Number(s): Table 2

Revised Text: N/A

8. Why did the photon/second/cm3/sr at Day 14 was reduced from Day 7 (Table 1) needs to be explained.

Response: WE thank the reviewers for pointing this out and have addressed it in the text.

Line Number(s): 249-252

Revised Text: At Day 14 it was noted (Table 1) that the photon/second/cm3/sr read was decreasing and then increased at subsequent timepoints as expected. This speaks to the high variance of IVIS at early timepoints, and why having an added system of tumor progression analysis, such as TAUS, is essential.

9. Tables 1-2 should have titles.

Response: Titles have been added.

Line Number(s): Table 1/2

Revised Text: N/A

Reviewer #2:

1. IVIS 2D imaging, 15 sec exposure seems long even for the earlier time points. Did the author try different time of exposure?

Response: The 2D IVIS system has an automatic exposure setting of 0.5-60seconds determined by the initially snapshots collected to maintain the signal within the linear range and prevent oversaturation. To account for this, before analysis all images were manually adjusted to the same minimum and maximum luminescent scales, as standard manufacturer operating procedure details.

Line Number(s): N/A

Revised Text:N/A

2. Table 1-under IVIS imaging, the right part of the table, the intensity should be shown as cm2 if it’s 2D imaging.

Response: This has been amended.

Line Number(s): Table 1

Revised Text: N/A

3. Tables 1 and 2- what does the n=20 mean under “Transabdominal ultrasound measurement”

Response: For each timepoint, when each individual mouse underwent ultrasound, the abdomen was assessed for tumor in four locations based on abdominal quadrants: 1) right upper, right lower, left upper, left lower. In order to provide specific information about tumor size discrimination with ultrasound, we reported the individual size of each tumor implant. This has been further clarified within the methods.

Line Number(s): 174-178

Revised Text: “For each timepoint, when each individual mouse underwent ultrasound, the abdomen was assessed for tumor in four locations based on abdominal quadrants: 1) right upper, 2) right lower, 3) left upper, 4) left lower. In order to provide specific information about tumor size discrimination with ultrasound, the individual size of each tumor implant was reported.”

4. Figure 3, image from day 7 is missing and should be included.

Response: This has been added to the figure.

Line Number(s): N/A

Revised Text:Figure 3

5. Consider including a figure illustrating the study design

Response: A figure explaining study design has been added.

Line Number(s): Figure 1

Revised Text: Figure 1

6. Provide more details about the PDX and the PDX study- patient information, tumor information, number of cells injected, number of mice injected and any other time points assessed using TAUS or was only day 10. Did ref 27 also examine pre-necropsy time points?

Response: Imaging of mice with PDX tumors were performed as a pilot study to demonstrate whether tumors could be detected. Details of this have been expanded in the methods section.

Line Number(s): 207-213.

Revised Text: “In order to assess the utility of TAUS in PDX models of EOC, the investigators performed TAUS in the manner described above in a small cohort of mice (n=5). In this pilot experiment, tumor samples were obtained at the time of primary debulking surgery for high grade serous EOC or uterine serous carcinoma, processed into a single-cell suspension and injected via IP injection. TAUS was done at four time points – 3 days, 10 days, 14 days and 21 days post-IP injection.”

7. Are there other IVIS imaging studies using the ID8-luc model that the authors can directly compare their results at similar time points?

Response: Our bioluminescence results for IVIS are comparable to those reported in Liao et al [21] utilizing ID8-luciferase cells with both studies detecting tumor growth at 2 weeks post-injection. In their study, they reported that bioluminescence imaging was able to detect tumor growth and engraftment earlier than performing mouse weight measurements, alone.

Line Number(s): 262-264

Revised Text: “Our findings are consistent with prior studies demonstrating that IVIS is able to detect tumor growth at 2 weeks after ID8-luc cell injection [21].”

8. Line 66: IVIS not IVUS

Response: This changed has been made.

Line Number(s): Line 66

Revised Text: “TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVIS.”

Submitted filename: Plos One Response to Reviewers [Apr 01 2020].docx

Click here for additional data file.

10 Apr 2020

Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer

PONE-D-20-00914R1

Dear Dr. Reizes,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Shannon M. Hawkins, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors addressed previous concerns by including additional data and requested information.

I am satisfied with the revised version.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

15 Apr 2020

PONE-D-20-00914R1

Use of Transabdominal Ultrasound for the Detection of Intra-Peritoneal Tumor Engraftment and Growth in Mouse Xenografts of Epithelial Ovarian Cancer

Dear Dr. Reizes:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Shannon M. Hawkins

Academic Editor

PLOS ONE