Short communication: Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry.

BACKGROUND: Parotid cancer is relatively rare, and malignancy varies; therefore, novel markers are needed to predict prognosis. Recent advances in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), useful for visualization of lipid molecules, have revealed the relationship between cancer and lipid metabolism, indicating the potential of lipids as biomarkers. However, the distribution and importance of phospholipids in parotid cancer remain unclear.
OBJECTIVE: This study aimed to use MALDI-IMS to comprehensively investigate the spatial distribution of phospholipids characteristically expressed in human parotid cancer tissues.
METHODS: Tissue samples were surgically collected from two patients with parotid cancer (acinic cell carcinoma and mucoepidermoid carcinoma). Frozen sections of the samples were assessed using MALDI-IMS in both positive and negative ion modes, with an m/z range of 600-1000. The mass spectra obtained in the tumor and non-tumor regions were compared and analyzed. Ion images corresponding to the peak characteristics of the tumor regions were visualized.
RESULTS: Several candidate phospholipids with significantly different expression levels were detected between the tumor and non-tumor regions. The number of unique lipid peaks with significantly different intensities between the tumor and non-tumor regions was 95 and 85 for Cases 1 and 2, respectively, in positive ion mode, and 99 and 97 for Cases 1 and 2, respectively, in negative ion mode. Imaging differentiated the characteristics that phospholipids were heterogeneously distributed in the tumor regions.
CONCLUSION: Phospholipid candidates that are characteristically expressed in human parotid cancer tissues were found, demonstrating the localization of their expression. These findings are notable for further investigation of alterations in lipid metabolism of parotid cancer and may have potential for the development of phospholipids as biomarkers.

Introduction

Parotid cancer, the most common salivary gland cancer, has various histological types and grades of malignancy [1, 2]. Because of its clinical and histological variety, it is difficult to estimate the malignancy grade and histological type preoperatively [3]. Therefore, not only the improvement of clinical and pathological diagnosis, but also prognostic estimation methods for parotid cancer are necessary to provide appropriate treatment according to the grade of malignancy. In addition, the carcinogenic mechanism of parotid cancer remains unclear because of its rarity and variety of histological types. Although specific genes and proteins have been evaluated as practical markers for parotid cancer, lipid molecules have not been investigated to date [4-6].

Lipids play an important role in various biological functions [7]. In recent years, the role of lipid molecules in biological functions has been clarified, and the importance of the relationship between cancer and lipid metabolism has attracted much attention [8-10]. Since lipid metabolism affects cellular processes, such as cell growth and division, and is associated with carcinogenesis, it has been suggested that phospholipid-related compounds may be new biomarkers [11].

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) technology has recently been advanced to investigate the distribution of phospholipid expression [12]. Compared to conventional methods that require labeling, mass spectrometry (MS) allows the analysis of a wide variety of molecules, with only minor structural differences and without the need for labeling. In addition, imaging mass spectrometry (IMS) enables visualization of the distribution of many biomolecules by overlaying microscopic images and mass spectrometric data for image analysis [13, 14]. MALDI-IMS has been used to study the localization of proteins and phospholipids in some cancer tissues [15, 16]. In preceding studies, the expression patterns of specific phospholipids have been reported to differ between cancerous and non-cancerous regions in adenocarcinomas such as prostate and breast cancers [17, 18]. Furthermore, it has been suggested that these phospholipids may be potential biomarkers [15, 19, 20]. Therefore, it is possible that the expression of phospholipids is also a characteristic of the difference between cancerous and non-cancerous regions in parotid cancer, but there have been no reports on phospholipids in parotid cancer.

In this study, a MALDI-IMS-based lipidomic strategy was employed to profile the differentially expressed candidate phospholipids between parotid cancer tissues and the corresponding adjacent non-cancerous salivary gland tissues as a first step toward finding the importance of phospholipids in parotid cancer.

Materials and methods

Ethics statement

The present study was conducted in accordance with the Declaration of Helsinki and its latest amendments, and was approved by the Ethics Committee of Osaka Medical College (approval no. 2020–211). Written informed consent was obtained from all participants.

Sample collection

Tissue samples were collected from surgically removed tissues of two Japanese patients who underwent surgery with a diagnosis of parotid cancer at Osaka Medical College Hospital, Takatsuki City, Japan, in 2020. The patients were not previously diagnosed and had not received any prior treatment. They were both male, 32 and 65 years of age. The histologic type of cancer tissue was acinic cell carcinoma and mucoepidermoid carcinoma, and both were low- to intermediate-grade malignancies. Tissue samples were cut into tissue blocks immediately after surgical removal (S1 Fig), frozen in isopentane cooled to -80°C with dry ice, and stored in a -80°C freezer until analysis.

Tissue section preparation

The tissue blocks were sliced to a thickness of 10 μm at -20°C using a cryostat (CM1950; Leica, Wetzler, Germany). Serial tissue sections were mounted on an indium tin oxide-coated glass slide (Bruker Daltonics, Bremen, Germany) and a Matsunami adhesive silane-coated glass slide (Matsunami, Osaka, Japan) for IMS analysis and hematoxylin and eosin (HE) staining, respectively. Before analysis by IMS, the pathologist confirmed that the tumor and non-tumor regions of parotid cancer were present in a single section using HE-stained images of serial tissue sections. The non-tumor areas confirmed to be normal parotid tissue were included in the analysis. Regions with insufficient pathological findings were excluded from the analysis.

For IMS, each tissue section was coated with 9-aminoacridine (Merck, Darmstadt, Germany), which served as the matrix. Each slide was coated with a 9-aminoacridine matrix layer obtained by sublimation at 220°C, and the thickness was set to 1 μm using IMLayer (Shimadzu Corporation, Kyoto, Japan).

IMS analysis

The tissue sections were analyzed using an imaging mass microscope (iMScope TRIO, Shimadzu Corporation, Kyoto, Japan) equipped with a 355-nm Nd: YAG laser, which has a mass resolution of 10,000 and a mass accuracy of less than 20 ppm. Mass spectrometry data were acquired in positive and negative ion modes in the 600.0–1000.0 m/z range using an external calibration method. The interval between each data point was 50 μm. The mass spectrometry parameters were manually optimized to obtain the highest sensitivity.

IMS data analysis

Imaging MS Solution version 1.12.26 (Shimadzu Corporation, Kyoto, Japan) was used to normalize the mass data to the total ion current and eliminate variations in ionization efficiency. A total of 100 spectra with strong average intensities were extracted, and hierarchical cluster analysis was performed using the Euclidean distance analysis method for inter-individual distances and Ward’s method for cluster distances. Of the cluster images obtained by hierarchical cluster analysis, mass spectrometry images of the masses specifically detected in the tissue were created. The phospholipids analyzed were phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidic acid (PA), along with sphingomyelin (SM). Candidate compounds with masses (m/z) consistent with these theoretical masses (assumed to be [M+H]+ or [M-H]-) were estimated from the Human Metabolome Database (https://hmdb.ca/spectra/ms/search).

Based on the results of the HE staining of serial sections, regions of interest (ROIs) were defined for parotid cancer areas and non-tumor areas (S1C and S1D Fig). The signal intensities of each defined ROI were statistically compared using the Welch’s t-test. A p-value of < 0.01 was set to be statistically significant.

Results

Histopathological findings

The pathological diagnosis of Case 1 was acinic cell carcinoma, and that of Case 2 was mucoepidermoid carcinoma. Both were cases of locally advanced intermediate-grade cancer. The tissue removed during surgery in Case 1 was approximately 4 cm in diameter, and the cut surface of the tumor was yellowish-brown and cystic (S1A Fig). HE staining in Case 1 revealed serous glandular cell-like tumor cells with hematoxylin-stained basophilic granules in the cytoplasm, which proliferated in a solid pattern (S1C Fig). In Case 2, the tumor was approximately 4 cm in diameter, the cut surface was bifid, and numerous cystic spaces filled with mucus were observed (S1B Fig). The enlarged image revealed presence of mucous cells, epidermoid cells, and intermediate cells (S1D Fig). The ROI areas of the tissue corresponding to the tumor and non-tumor areas were determined by an experienced pathologist.

Differences in mass spectra of tumor and non-tumor region

Two parotid cancer tissue specimens from the two cases were analyzed by IMS. Those contained tumor and non-tumor regions in the same section. The average mass spectra obtained from the tumor and non-tumor ROIs determined by HE staining in S1 Fig are shown in S2 and S3 Figs. The number of peaks with significantly different intensities (Welch’s t-test, p < 0.01) between the two ROIs were 95 and 99 in positive and negative ion modes for Case 1, respectively, and 85 and 97 in positive and negative ion modes for Case 2, respectively.

Among these, altered m/z values for which the ratio of the average spectral intensities between ROIs was more than double, and the median value of spectral intensities is more than 200 in the positive ion mode, identified by IMS in positive ion mode and negative ion mode were shown in Tables 1 and 2, respectively. The numbers of m/z common in Case 1 and 2 in the positive ion mode were three (m/z 705.58, 706.53, and 725.56) with increased expression in tumors and seven (m/z 786.61, 787.62, 788.61, 952.66, 953.66, 954.68, and 980.70) with decreased expression in tumors (Table 1); whereas, in the negative ion mode, there were five (m/z 616.45, 630.46, 642.46, 659.16, and 661.46) with increased expression in tumors and four (m/z 770.51, 786.50, 861.52, and 862.52) with decreased expression in tumors (Table 2).

Table 1

Altered peaks identified by MALDI-IMS and corresponding candidate phospholipids in positive ion mode.

A. Up-regulated in Tumor
Case 1							Case 2
m/z	ratioa	Candidate phospholipids					m/z		ratioa	Candidate phospholipids
706.53	4.3	PC(30:0)		PE(33:0)			666.49		3.2	-
725.56	3.4	PA(38:4)					697.48		3.2	PA(34:1)	PA(36:4)	PS(28:0)
726.56	3.2	PE(36:3)*								PE(33:4)	PG(30:2)
741.53	2.9	-					667.50		3.0	PG(28:0)
703.59	2.6	SM(34:1)					727.58		2.7	-
704.59	2.5	PC(31:0)*		PE(34:1)*		PE(34:0)*	706.56		2.4	PC(30:0)	PE(33:0)
720.58	2.4	PC(31:0)		PE(34:0)			698.48		2.3	PE(33:4)	PE(34:3)*
705.58	2.4	SM(34:0)		PA(36:0)			694.47		2.2	PS(29:0)
734.57	2.2	PC(32:0)		PE(35:0)			705.60		2.2	SM(34:0)
735.57	2.1	-					688.41		2.1	-
							725.56		2.0	PA(38:4)
B. Down-regulated in Tumor
Case 1							Case 2
m/z	ratioa	Candidate phospholipids					m/z	ratioa		Candidate phospholipids
860.53	0.2	PS(42:8)					796.54	0.2		PS(37:5)	PE(40:4)	PC(37:4)
980.70	0.3	-					759.58	0.3		PA(40:1)
786.61	0.4	PC(36:2)	PE(40:1)*				953.66	0.3		-
953.66	0.4	-					952.66	0.3		-
787.62	0.4	PA(42:1)	SM(38:1)				798.56	0.3		PS(37:4)	PE(40:3)
952.66	0.4	-					785.60	0.4		PA(42:2)
955.68	0.4	-					786.61	0.4		PC(36:2)	PE(40:1)*
954.68	0.4	-					784.59	0.4		PC(36:3)	PE(40:2)*
788.61	0.5	PE(39:1)	PC(36:1)		PE(40:0)*		787.62	0.4		PA(42:1)
675.47	0.5	PA(34:1)					758.59	0.4		PE(37:2)	PC(34:2)	PE(38:1)*
							780.55	0.4		PC(36:5)	PE(40:4)*
							804.55	0.4		PC(38:7)	PS(36:1)
							781.56	0.4		PA(42:4)
							806.57	0.4		PC(38:6)	PS(36:0)
							807.57	0.4		PA(44:5)	PG(38:0)
							980.70	0.4		-
							731.61	0.4		SM(36:1)
							783.58	0.5		PA(42:3)
							788.61	0.5		PE(39:1)	PC(36:1)	PE(40:0)*
							782.57	0.5		PC(36:4)	PE(40:3)*
							760.60	0.5		PC(34:1)	PE(37:1)	PE(38:0)*
							761.60	0.5		PA(40:0)
							808.59	0.5		PC(38:5)
							809.59	0.5		PA(44:4)
							789.62	0.5		PA(42:0)
							954.68	0.5		-

The list of m/z, where the mean spectrum is significantly different between tumor and non-tumor regions (Welch’s t-test, p < 0.01) and the ratio of mean spectral intensities is more than double, and the median value of spectral intensities is more than 200. The candidate phospholipids estimated from the Human Metabolome Database (https://hmdb.ca/spectra/ms/search)corresponding to these m/z values are presented. The m/z common to Case 1 and Case 2 is shown by shading.

a The ratio of relative signal intensity of tumor region to non-tumor region.

*deoxidation products. PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidylserine, PG: phosphatidylglycerol, PA: phosphatidic acid, and SM: sphingomyelin.

Table 2

Altered peaks identified by MALDI-IMS and corresponding candidate phospholipids in negative ion mode.

A. Upregulated in Tumor
Case 1					Case 2
m/z	ratioa	Candidate phospholipids			m/z	ratioa	Candidate phospholipids
809.48	9.7	PI (32:0)			661.46	3.1	PA(33:0)
630.46	3.5	PE(28:2)			659.20	2.9	-
661.46	3.4	PA(33:0)			630.46	2.8	PE(28:2)
642.46	3.3	-			644.48	2.6	PE(30:1) *
659.16	3.2	-			631.47	2.5	-
810.50	3.0	PE(42:10)	PS(38:4)		647.45	2.4	PA(32:0)
616.45	2.5	-			642.46	2.3	-
837.53	2.0	PI(34:0)			643.47	2.2	PA(32:2)
		-			616.45	2.1	-
					660.20	2.0	-
					718.51	2.0	PC(31:0)	PE(34:0)	PS(31:1)
B. Downregulated in Tumor
Case 1					Case 2
m/z	ratioa	Candidate phospholipids			m/z	ratioa	Candidate phospholipids
786.50	0.3	PE(40:8)	PS(36:2)		695.45	0.2	PA(36:4)
778.51	0.3	PE(40:7)	PS(36:1)	PC(38:6)	696.45	0.3	PE(33:4)
862.52	0.3	PS(42:6)			859.51	0.3	PI (36:3)
861.52	0.3	PI(36:2)			786.50	0.3	PE(40:8)	PS(36:2)
776.50	0.3	PC(36:6)	PS(36:7)	PS(35:0)	835.50	0.3	PI (34:1)
728.52	0.4	PC(32:2)	PE(37:2)	PE(36:1) *	836.52	0.3	PE(44:11)	PS(40:5)
		PC(33:1)*			770.51	0.3	PS(35:3)
771.51	0.4	PA(42:8)	PG(36:3)		834.48	0.4	PE(44:12)	PS(40:6)
770.51	0.4	PS(35:3)			774.52	0.4	PC(36:7)	PS(35:1)	PE(40:6)*
772.51	0.4	PS(35:2)	PC(36:8)	PE(40:7) *	747.47	0.4	PA(40:6)	PG(34:1)
773.51	0.4	PA(42:7)			790.53	0.4	PC(37:6)	PE(40:6)	PS(36:0)
885.53	0.5	PI (36:4)			861.52	0.4	PI (36:2)
886.52	0.5	PS(44:8)			883.49	0.4	PI (38:5)
727.49	0.5	PA(38:2)			862.52	0.4	PS(42:6)
726.50	0.5	PE(35:3)	PC(32:3)	PE(36:2) *	884.51	0.4	PS(44:9)
					788.53	0.4	PE(40:7)	PS(36:1)	PC(38:6) *
					789.51	0.4	-
					721.47	0.4	PA(38:5)	PG(32:0)
					863.52	0.4	PI (36:1)
					750.51	0.4	PC(34:5)	PE(37:5)	PE(38:4) *
					833.50	0.4	PI (34:2)
					764.48	0.5	PE(38:5)	PC(35:5)
					697.45	0.5	-
					740.51	0.5	PC(33:3)	PE(36:3)
					748.48	0.5	PE(37:6)	PC(34:6)	PS(33:0)
					857.48	0.5	PI (36:4)
					751.50	0.5	PE(37:6)
					794.51	0.5	PS(37:5)

The list of m/z, where the mean spectrum is significantly different between tumor and non-tumor regions (Welch’s t-test, p < 0.01) and the ratio of mean spectral intensities is more than double, and the median value of spectral intensities is more than 200. The candidate phospholipids estimated from the Human Metabolome Database (https://hmdb.ca/spectra/ms/search) corresponding to these m/z values are presented. The m/z common to Case 1 and Case 2 is shown by shading.

a The ratio of relative signal intensity of tumor region to non-tumor region.

*deoxidation products. PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidylserine, PG: phosphatidylglycerol, PA: phosphatidic acid, and SM: sphingomyelin.

Visualization by IMS of phospholipid candidates in human parotid cancer tissues

Among the images of each m/z obtained by IMS, Figs 1A and 2A show representative images of peaks (m/z) with significantly different signal intensities in the tumor and non-tumor areas, which are indicated by red and blue circles on the tumor/non-tumor dot graph in S2 and S3 Figs. Each m/z clearly distinguished the difference between tumor and non-tumor areas. Its expression was observed to be heterogeneously distributed within the tumor and non-tumor areas, respectively. The signal intensities of the tumor area (T) and non-tumor area (NT) at each m/z were significantly different (Welch’s t-test, p < 0.01) (Figs 1B and 2B).

Fig 1

Visualization by positive ion mode IMS of molecular distributions.

Hematoxylin and eosin (HE) stained images show defined regions of interest (ROIs) of tumor (T) and non-tumor (NT) areas. A. Representative images of peaks (m/z) with significantly different signal intensities in the tumor and non-tumor areas, which are indicated by red and blue circles on the tumor/non-tumor dot graph in S2 Fig. The threshold of the color scale was adjusted for each ion image to show a clear distribution. B. Box plots represent the signal intensities of the T and NT areas at each m/z. The significance of the difference of signal intensity between tumor and non-tumor areas was determined using Welch’s t-test. A p-value of less than 0.01 was obtained for all m/z signals compared here.

Fig 2

Visualization by negative ion mode IMS of molecular distributions.

Hematoxylin and eosin (HE) stained images show defined tumor (T) and non-tumor (NT) areas. A. Representative images of peaks (m/z) with significantly different ion intensities in the tumor and non-tumor areas, which are indicated by red and blue circles on the tumor/non-tumor graph in S3 Fig. The threshold of the color scale was adjusted for each ion image to show a clear distribution. B. Box plots represent the signal intensities of the T and NT regions at each m/z. The significance of the difference between tumor and non-tumor regions was determined using Welch’s t-test. A p-value of less than 0.01, was obtained for all m/z signals compared here.

Phospholipid candidates characteristic of human parotid cancer tissues

The candidate phospholipids corresponding to altered peaks identified by IMS were estimated from the database. The number of phospholipid candidates in the positive mode that were upregulated in tumor area were: 4, PC; 7, PE; 0, PI; 2, PS; 2, PG; 3, PA; and 2, SM; downregulated in tumor area were 11, PC; 9, PE; 0, PI; 5, PS; 0, PG; 10, PA; and 2, SM (Table 1). As for the negative mode, the number of phospholipid candidates that were upregulated in tumor area were: 1, PC; 4, PE; 2, PI; 2, PS; 0, PG; 2, PA; and 0, SM; downregulated in tumor area were 12, PC; 15, PE; 8, PI; 15, PS; 3, PG; 7, PA; and 0, SM (Table 2).

Discussion

In this study, the lipid distribution in human parotid cancer tissues was analyzed using MALDI-IMS, which identified several candidate phospholipids whose expression levels differed between tumor and non-tumor regions. This suggests that parotid cancer tissue has a markedly different phospholipid composition compared to that of non-tumor tissue. Alterations in the lipid composition of tissues have been reported in several cancers, including breast cancer [18, 21], prostate cancer [17, 19], lung cancer [22], kidney cancer [23], pharyngeal cancer [24], and oral cancer [20, 25, 26]. Furthermore, it has been suggested that these specific lipid profiles vary from among carcinomas, and may be potential diagnostic, prognostic, and predictive biomarkers. These facts suggest that it is necessary to individually examine each tumor. Therefore, it is worthwhile to further investigate the changes in the lipid composition in parotid cancer.

Molecular visualization has traditionally been important in the characterization of clinical specimens of parotid cancer. For example, immunohistochemical staining was performed for visualization of the expression of protein of myoepithelial and basal cell markers, such as alpha-smooth muscle actin, calponin, p63, and S100 [27, 28], and receptors, such as HER2 [29] and androgen receptors. In addition, fluorescence in situ hybridization is used for the detection of fusion genes, such as the ETV6-NTRK3 fusion gene in secretory carcinoma and the CRTC1/3-MAML2 fusion gene in mucoepidermoid carcinoma [3, 30]. In this study, MALDI-IMS was used to visualize lipid molecules in clinical parotid cancer specimens. MALDI-IMS has been used to investigate the localization of phospholipids in some cancer tissues [15], as the information from mass spectrometry, together with their location in the sample, can be obtained simultaneously. The results of this study show for the first time that MALDI-IMS may be useful as a new method for the clinical study of parotid cancer.

Here, MALDI-IMS analysis was performed in both positive and negative ion modes to comprehensively evaluate a large number of molecules. It is known that the positive ion mode is excellent for detecting PC and SM, and the negative ion mode is suitable for detecting PI, PS, and PG [31]; PE is detected in both modes [32]. In this study, there was a tendency to detect more PC and SM in positive ion mode, while more PI and PG were detected in the negative ion mode as phospholipid candidates whose expression levels differed significantly between tumor and non-tumor regions. In addition, PI was not detected in the positive ion mode, and SM was not detected in the negative ion mode. These results are consistent with the characteristics of the analytical method and suggest that further detailed analyses should be carried out, considering the characteristics of the analytical method. Furthermore, in this study, we used 9-aminoacridine as a matrix, which is one of the most commonly used MALDI matrices for lipid analysis, because it can ionize target molecules in both positive and negative ion modes [33]. Since the analytical sensitivity of lipid imaging depends on the type of matrix [34], the choice of matrix should also be considered in future analyses.

The present study suggests that phospholipid composition is significantly different between tumor and non-tumor regions of parotid cancer. The differences in the profiles of these phospholipids may be related to the well-known features of cancer cells. In general, rapidly proliferating cancer cells are characterized by enhanced lipid synthesis and changes in the composition of the cell membrane, which may be associated with an increase in PC and saturated phospholipids [8, 9]. The number of lipid rafts involved in cell signaling is increased in cancer cells, which may be associated with increased cholesterol and SM [35]. In addition, lipids, such as free fatty acids and prostaglandins, are involved in the communication between cancer and stromal cells. Therefore, it is possible that these changes in the cancer cells of the parotid cancer lesions in the present study were due to differences in lipid profiles. The phospholipid candidates common to the two cases of different histological types in this study are likely to be related to the characteristics of the cancer cells. In contrast, for phospholipid candidates whose expression is upregulated in a particular tissue type, it may be characteristic of that tissue type. Since the present study was based only on two cases of parotid cancer, further analysis with a larger number of cases is needed to clarify these possibilities.

Although the present results have revealed novel insights into the distribution of lipids in parotid cancer, this study should also be considered in the context of certain limitations. First, the design of this study aimed to confirm the distribution of the compound in the tissue and not to identify the compound. In this study, we estimated candidate phospholipids from a database based on mass peaks of compounds differentially expressed in tumor and non-tumor areas of parotid cancer tissues. We believe that other methods, such as LC MS/MS analysis [36] of parotid cancer tissue, should be used to identify lipid molecules that are differentially expressed in tumor and non-tumor tissues. Therefore, we plan to perform additional experiments and report those findings in a separate study. Second, only two cases of parotid cancer were included in this study, which is not sufficient to clarify the characteristics of parotid cancer. Since parotid cancer has diverse clinical and histological characteristics, such as age and sex, it is necessary to analyze a larger number of cases in order to clarify the effects of these factors on phospholipid expression. Nevertheless, this is a preliminary pilot study with a limited sample size, and the main aim was to demonstrate that the lipid profile of parotid cancer tissue is a characteristic of the tumor lesion. In this context, this study is a promising proof of concept and opens up a new pathway for the use of lipid profiles as a potential diagnostic tool for parotid cancer. These limitations need to be addressed in future research.

Conclusions

The lipid distribution in human parotid cancer tissues was analyzed using MALDI-IMS, and candidate phospholipids differentially expressed in tumor and non-tumor areas were profiled. Further investigation of changes in lipid metabolism in parotid cancer is worthwhile.

Parotid gland cancer tissue.

Parotid gland cancer tissue removed during surgery and cut out tissue in Cases 1 (A) and 2 (B). Scale bar: 1.0 cm. HE-stained tissue of Cases 1 (C) and 2 (D). Scale bar: 500 μm; enlarged images: 50 μm. The yellow dashed area indicates ROI of tumor areas, and the blue dashed area indicates ROI of non-tumor areas. HE, hematoxylin and eosin; ROI, region of interest.

(TIF)

Click here for additional data file.

Mass spectra and relative intensity ratios of tumor and non-tumor areas in positive ion modes.

The spectra in the mass range of m/z 600–1000 in positive ion modes are shown for the tumor and non-tumor regions distinguished in S1 Fig of Case 1 and Case 2. The horizontal axis shows m/z, and the vertical axis shows the relative intensity. The dot graph of the ratio of relative intensities of tumor and non-tumor regions at each m/z is shown below. Of the peaks (m/z) with a median value of spectral intensity greater than 200, red circles indicate the top six m/z with a higher expression ratio and blue circles indicate the top six m/z with a lower expression ratio in tumor areas compared to non-tumor regions. Fig 1 shows the IMS images of each of these m/z values.

(TIF)

Click here for additional data file.

Mass spectra and relative intensity ratios of tumor and non-tumor areas in negative ion modes.

The spectra in the mass range of m/z 600–1000 in negative ion modes are shown for the tumor and non-tumor regions distinguished in S1 Fig of Case 1 and Case 2. The horizontal axis shows m/z, and the vertical axis shows the relative intensity. The dot graph of the ratio of relative intensities of tumor and non-tumor regions at each m/z is shown below. Of the peaks (m/z) with the median value of spectral intensity greater than 200, red circles indicate the top six m/z with a higher expression ratio and blue circles indicate the top six m/z with a lower expression ratio in tumor regions compared to non-tumor regions. Fig 2 shows the IMS images of each of these m/z values.

(TIF)

Click here for additional data file.

6 Oct 2021

PONE-D-21-27808Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometryPLOS ONE

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I am pleased to inform you that PLOS ONE has found that your manuscript entitled: " Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry; Manuscript Number PONE-D-21-27808" worth publishing.

However, according to the one referee, your manuscript needs some corrections and needs major revision

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Major comment:

The paper tackles and interesting subject and uses IMS to profile lipids for Parotid cancer, an absolutely novel idea. However, I suggest that the paper is re-written and significantly shorten as it is more suitable for a “short report” rather than being a full research article. The authors rightfully acknowledge the main limitation of the study that is the sample size and with only 2 samples for rather different cancer type, the paper is only suitable for a short report or short communication-type paper. It will require significant reduction and re-writing

Additional comments:

• The instrument type needs to be described including its resolution and mass error. I see m/z values reported with one significant digit. That is not suitable for discovery-based paper like this one.

• Putative identification is needed; hence the importance of the mass accuracy

• Why the authors did not do MSMS

• What normalization strategy was used – did they use matrix peaks for that

• The two patients and males within very different age groups- is this a concern?

• Why 9-aminoacridine is used for both positive and negative mode analysis. Matrix choice will have a profound effect on IMS results

Reviewer #2: PONE-D-21-27808

This work focuses on the identification of phospholipids biomarkers for parotid cancer. These biomarkers were identified by comparing the lipid profiles of the tumor and non-tumor tissue by MALDI imaging. Consequently, several lipid peaks were significantly regulated (down- or Up-regulated) due to the parotid cancer. This pilot study is a straightforward biomarker identification using comparative lipidomics, and it opens a way for further future studies on the alterations in lipid metabolism of parotid cancer.

Overall, I recommend the publication of this work. Also, I recommend adding a separate conclusion section at the end.

**********

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13 Nov 2021

November 12, 2021.

Dr. Joseph Banoub

Academic Editor,

PLOS ONE

Dear Dr. Joseph Banoub,

Thank you for your comments regarding our paper (Manuscript No: Ms. PONE-D-21-27808) entitled “Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry” by Kanetake H. et al. for publication in PLOS ONE. We have modified our paper according to the reviewers’ comments. Our responses to each point raised by reviewers are as follows:

Reply to reviewer #1:

1. Concern of the reviewer: Major comment: The paper tackles and interesting subject and uses IMS to profile lipids for Parotid cancer, an absolutely novel idea. However, I suggest that the paper is re-written and significantly shorten as it is more suitable for a “short report” rather than being a full research article. The authors rightfully acknowledge the main limitation of the study that is the sample size and with only 2 samples for rather different cancer type, the paper is only suitable for a short report or short communication-type paper. It will require significant reduction and re-writing

Our response: Thank you for the time you spent reviewing our work. Because of the relatively low incidence of parotid cancer, it takes years to perform studies with large sample sizes. Therefore, as the reviewer points out, it is worth reporting the results of this sample size as a "short report". Accordingly, we have shortened the manuscript by 294 words to a revised total of 2615 words. This has been achieved by changing the style of expression and deleting unnecessary text. In addition, three Figures have been adapted to supplementary materials. Furthermore, the title has been revised. Please refer to these in the revised version.

2. Concern of the reviewer: Additional comments: The instrument type needs to be described including its resolution and mass error. I see m/z values reported with one significant digit. That is not suitable for discovery-based paper like this one.

Our response: As the reviewer pointed out, the resolution and mass accuracy of the mass spectrometer are important in the interpretation of the results. Therefore, the specifications of the imaging mass microscope (iMScope TRIO) used in this study, with a mass resolution of 10,000 and a mass accuracy of less than 20 ppm, have been added to the Materials and methods section (p.7, lines 126–127). Additionally, in accordance with the reviewers' instructions regarding the notation of m/z values, all m/z values have been corrected to two significant digits.

Revised text: p.7, lines 126–127; The tissue sections were analyzed using an imaging mass microscope (iMScope TRIO, Shimadzu Corporation, Kyoto, Japan) equipped with a 355-nm Nd: YAG laser, which has a mass resolution of 10,000 and a mass accuracy of less than 20 ppm.

3. Concern of the reviewer: Additional comments: Putative identification is needed; hence the importance of the mass accuracy.

Our response: As indicated by the reviewer, we also think that the putative identification of compounds would be valuable. In this study, we estimated candidate phospholipids from a database based on mass peaks of compounds differentially expressed in tumor and non-tumor areas of parotid cancer tissues. For more accurate identification of candidate phospholipids, other experiments, such as MS/MS, are required. However, the present study aimed to determine the distribution of the phospholipids in parotid cancer tissue, and we consider this paper to be a report of a preliminary study. The results of this study suggest that there may be phospholipids with different distribution patterns in tumor and non-tumor areas, which will be identified in more detail and presented in a separate study. To make this point clearer, we have added the following statements to the Discussion section.

Revised text: p.17, line 324–p.18, line 331; First, the design of this study aimed to confirm the distribution of the compound in the tissue and not to identify the compound. In this study, we estimated candidate phospholipids from a database based on mass peaks of compounds differentially expressed in tumor and non-tumor areas of parotid cancer tissues. We believe that other methods, such as LC MS/MS analysis [34] of parotid cancer tissue, should be used to identify lipid molecules that are differentially expressed in tumor and non-tumor tissues. Therefore, we plan to perform additional experiments and report those findings in a separate study.

4. Concern of the reviewer: Additional comments: Why the authors did not do MSMS.

Our response: The remarks of the reviewer are very valuable. We have included additional details on this issue in the “Discussion” section (p.17, line 324–p.18, line 331). As described above, the present study focuses on investigating the distributional localization of phospholipid expression in parotid cancer tissues to determine whether there are phospholipids differentially expressed between tumor and non-tumor areas. Since the results of this study suggest this possibility, the next step should be the identification of candidate phospholipids. In this study, the candidate phospholipids were estimated from the database based on the peak mass by IMS. As the reviewer pointed out, MS/MS is very valuable for validation and identification of the obtained presumed phospholipids. However, in the present case, it was difficult to collect appropriate samples from tumor and non-tumor areas for MS/MS analysis. Furthermore, parotid cancer is a rare disease, and it is not easy to collect a large number of cases. Therefore, as a next step, we plan to increase the number of parotid carcinoma cases, identify phospholipids that are differentially expressed between tumor and non-tumor areas, and report those results in a separate study in the near future.

Revised text: p.17, line 324–p.18, line 331; First, the design of this study aimed to confirm the distribution of the compound in the tissue and not to identify the compound. In this study, we estimated candidate phospholipids from a database based on mass peaks of compounds differentially expressed in tumor and non-tumor areas of parotid cancer tissues. We believe that other methods, such as LC MS/MS analysis [34] of parotid cancer tissue, should be used to identify lipid molecules that are differentially expressed in tumor and non-tumor tissues. Therefore, we plan to perform additional experiments and report those findings in a separate study.

5. Concern of the reviewer: Additional comments: What normalization strategy was used – did they use matrix peaks for that.

Our response: As described in the Materials and methods section (p.7, lines 134–136), in IMS data analysis, the acquired raw mass spectra were normalized by the total ion current (TIC) using Imaging MS Solution version 1.12.26 (Shimadzu Corporation). The matrix peaks were not used specifically for normalization. The variation in the ionization efficiency, which is caused by the heterogeneous distribution of matrix crystals and their sublimation during measurement, was eliminated for each data point by equalizing the TIC of each mass spectra. Normalization of spectra by TIC has been reported to improve IMS visualization quality (Sugiura Y, et al. J Lipid Res. 2009).

6. Concern of the reviewer: Additional comments: The two patients and males within very different age groups- is this a concern?

Our response: The two cases in this study are a 32-year-old man with acinic cell carcinoma and a 65-year-old man with mucoepidermoid carcinoma. Parotid gland cancer is a relatively rare lesion with a wide variation in biological and histological characteristics. Epidemiologically, there is a wide age range at diagnosis of parotid cancer. Therefore, this preliminarily study included acinic cell carcinoma and mucoepidermoid carcinoma, which are common histological types of parotid cancer, at different ages. Based on the results of this study, it is not possible to evaluate the results according to age, gender, or histological types. Further analysis of a larger number of cases will help to clarify the influence of these factors on phospholipid expression. The Discussion section (p.18, lines 331–335) has been revised to make these clearer.

Revised text: p.18, lines 331–335; Second, only two cases of parotid cancer were included in this study, which is not sufficient to clarify the characteristics of parotid cancer. In addition, because there are many histological types of parotid cancer, a larger number of cases should be analyzed to clarify the differences between each histological type. Since parotid cancer has diverse clinical and histological characteristics, such as age and sex, it is necessary to analyze a larger number of cases in order to clarify the effects of these factors on phospholipid expression.

7. Concern of the reviewer: Additional comments: Why 9-aminoacridine is used for both positive and negative mode analysis. Matrix choice will have a profound effect on IMS results.

Our response: 9-aminoacridine is one of the MALDI matrices commonly used for lipid analysis. The aim of this study was to comprehensively detect phospholipid compounds differentially expressed between tumor and non-tumor areas. Thus, we performed measurements in both modes since phospholipid compounds that are likely to be detected in positive and negative modes are different. 9-aminoacridine can ionize target molecules in both positive and negative ion modes and has been shown to be useful in the analysis of lipid imaging (Perry WJ, et al. J Mass Spectrom. 2020). Therefore, in the present study, 9-aminoacridine was used as a matrix in the first stage of IMS of phospholipids in parotid cancer. As pointed out by the reviewer, the analytical sensitivity of lipid imaging can vary when different types of matrices are employed (Tobias F, et al. J Proteome Res. 2020); thus, the choice of matrix should be considered a future issue. We have included additional details on this issue in the “Discussion” section (p. 16, lines 301–p.17. lines 305 ).

Revised text: p. 16, lines 301–p.17. lines 305; Furthermore, in this study, we used 9-aminoacridine as a matrix, which is one of the most commonly used MALDI matrices for lipid analysis, because it can ionize target molecules in both positive and negative ion modes (Perry WJ, et al. J Mass Spectrom. 2020). Since the analytical sensitivity of lipid imaging depends on the type of matrix (Tobias F, et al. J Proteome Res. 2020), the choice of matrix should also be considered in future analyses.

Reply to reviewer #2:

1. Concern of the reviewer: This work focuses on the identification of phospholipids biomarkers for parotid cancer. These biomarkers were identified by comparing the lipid profiles of the tumor and non-tumor tissue by MALDI imaging. Consequently, several lipid peaks were significantly regulated (down- or Up-regulated) due to the parotid cancer. This pilot study is a straightforward biomarker identification using comparative lipidomics, and it opens a way for further future studies on the alterations in lipid metabolism of parotid cancer.

Overall, I recommend the publication of this work. Also, I recommend adding a separate conclusion section at the end.

Our response: Thank you very much for the time you spent reviewing our work. In accordance with the reviewer's comment, we have added “Conclusions” section in the revised manuscript.

Revised text: p. 18, lines 342–346; Conclusions: The lipid distribution in human parotid cancer tissues was analyzed using MALDI-IMS, and candidate phospholipids differentially expressed in tumor and non-tumor areas were profiled. Further investigation of changes in lipid metabolism in parotid cancer is worthwhile.

Once again, we thank you for your kind suggestions and the time you have spent reviewing our work. We hope that you will find our revised manuscript suitable for publication in PLOS ONE.

Yours sincerely,

Hirofumi Kanetake

Department of Otorhinolaryngology-Head and Neck Surgery,

Osaka Medical and Pharmaceutical University.

E-mail: hirofumi.kanetake@ompu.ac.jp.

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

3 Dec 2021

Short communication: Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry

PONE-D-21-27808R1

Dear Dr. Kanetake,

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

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Kind regards,

Joseph Banoub, Ph,D., D. Sc., FCIC, FRSC

Academic Editor

PLOS ONE

Additional Editor Comments (optional):ALL

All queries  have been  answered

Reviewers' comments:

9 Dec 2021

PONE-D-21-27808R1

Short communication: Distribution of phospholipids in parotid cancer by matrix-assisted laser desorption/ionization imaging mass spectrometry

Dear Dr. Kanetake:

I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

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on behalf of

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