Spatial Transcriptomics Suggests That Alterations Occur in the Preneoplastic Breast Microenvironment of BRCA1/2 Mutation Carriers.

Breast cancer is the most common cancer in females, affecting one in every eight women and accounting for the majority of cancer-related deaths in women worldwide. Germline mutations in the BRCA1 and BRCA2 genes are significant risk factors for specific subtypes of breast cancer. BRCA1 mutations are associated with basal-like breast cancers, whereas BRCA2 mutations are associated with luminal-like disease. Defects in mammary epithelial cell differentiation have been previously recognized in germline BRCA1/2 mutation carriers even before cancer incidence. However, the underlying mechanism is largely unknown. Here, we employ spatial transcriptomics to investigate defects in mammary epithelial cell differentiation accompanied by distinct microenvironmental alterations in preneoplastic breast tissues from BRCA1/2 mutation carriers and normal breast tissues from noncarrier controls. We uncovered spatially defined receptor-ligand interactions in these tissues for the investigation of autocrine and paracrine signaling. We discovered that ß1-integrin-mediated autocrine signaling in BRCA2-deficient mammary epithelial cells may differ from BRCA1-deficient mammary epithelial cells. In addition, we found that the epithelial-to-stromal paracrine signaling in the breast tissues of BRCA1/2 mutation carriers is greater than in control tissues. More integrin-ligand pairs were differentially correlated in BRCA1/2-mutant breast tissues than noncarrier breast tissues with more integrin receptor-expressing stromal cells. IMPLICATIONS: These results suggest alterations in the communication between mammary epithelial cells and the microenvironment in BRCA1 and BRCA2 mutation carriers, laying the foundation for designing innovative breast cancer chemo-prevention strategies for high-risk patients.

Alterations in the preneoplastic breast microenvironment of BRCA1/2 mutation carriers revealed by spatial transcriptomics.

Breast cancer is the most common cancer in females, affecting one in every eight women and accounting for the majority of cancer-related deaths in women worldwide. Germline mutations in the BRCA1 and BRCA2 genes are significant risk factors for specific subtypes of breast cancer. BRCA1 mutations are associated with basal-like breast cancers, whereas BRCA2 mutations are associated with luminal-like disease. There are currently few chemoprevention strategies available for BRCA1/2 mutation carriers, and irreversible prophylactic mastectomy is the primary option. Designing chemo-preventive strategies requires an in-depth understanding of the physiological processes underlying tumor initiation. Here, we employ spatial transcriptomics to investigate defects in mammary epithelial cell differentiation accompanied by distinct microenvironmental alterations in preneoplastic breast tissues from BRCA1/2 mutation carriers and normal breast tissues from non-carrier controls. We uncovered spatially defined receptor-ligand interactions in these tissues for the investigation of autocrine and paracrine signaling. We discovered that ß1-integrin-mediated autocrine signaling in BRCA2-deficient mammary epithelial cells differs from BRCA1-deficient mammary epithelial cells. In addition, we found that the epithelial-to-stromal paracrine signaling in the breast tissues of BRCA1/2 mutation carriers is greater than in control tissues. More integrin-ligand pairs were differentially correlated in BRCA1/2-mutant breast tissues than non-carrier breast tissues with more integrin receptor-expressing stromal cells. These results reveal alterations in the communication between mammary epithelial cells and the microenvironment in BRCA1 and BRCA2 mutation carriers, laying the foundation for designing innovative breast cancer chemo-prevention strategies for high-risk patients.

Characterizing diversity among human oral stem-like cancer cells using flow cytometry.

We have developed the protocol of flow cytometry for characterizing diversity among oral stem-like cancer cells (SLCCs) using CD44, CD24, and aldehyde dehydrogenase (ALDH) in oral tumors. We are also reporting the protocol for tumor-derived explant cultures to develop oral cancer cell lines and enriching these diverse hybrid states of cancer cells in 3D spheroids from established cell lines. For complete details on the use and execution of this protocol, please refer to Vipparthi et al. (2022) and Vipparthi et al. (2021).

Emergence of hybrid states of stem-like cancer cells correlates with poor prognosis in oral cancer.

Cancer cell state transitions emerged as powerful mechanisms responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Here, by multiplexing phenotypic markers of stem-like cancer cells (SLCCs); CD44, CD24 and aldehyde dehydrogenase (ALDH), we characterized diversity among multiple oral tumor tissues and cell lines. Two distinct patterns of spontaneous transitions with stochastic bidirectional interconversions on 'ALDH-axis', and unidirectional non-interconvertible transitions on 'CD24-axis' were observed. Interestingly, plastic 'ALDH-axis' was harnessed by cells to adapt to a Cisplatin tolerant state. Furthermore, phenotype-specific RNA sequencing suggested the possible maintenance of intermediate hybrid cell states maintaining stemness within the differentiating subpopulations. Importantly, survival analysis with subpopulation-specific gene sets strongly suggested that cell-state transitions may drive non-genetic heterogeneity, resulting in poor prognosis. Therefore, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behavior in oral cancer; which may provide prerequisite knowledge for treatment strategies.

Two novel cell culture models of buccal mucosal oral cancer from patients with no risk-habits of tobacco smoking or chewing.

OBJECTIVE: Tobacco consumption is one of the major etiological factors for oral cancer, but it also develops in non-tobacco users, with unknown etiologies. Cellular models for tobacco associated oral cancer are available, however; reports of cellular models for studying non-tobacco associated oral cancer are limiting. We report here the establishment and characterization of two novel buccal mucosal cancer cell lines 'GBC02' and 'GBC035' derived from non-tobacco users. MATERIALS AND METHODS: Short tandem repeats (STR) profiling, Next-generation sequencing for whole-genome, exome and copy number alterations, immunofluorescence, flow-cytometry, proliferation, live-cell chemotaxis, 3D-spheroid formation, chemotherapy response, gene-expression microarray, gene-set enrichment analysis and xenograft development were performed. RESULTS: Sources of the established cultures were matched to their donors through STR profiling. Genome sequence analysis revealed somatic mutations in TP53, CASP8, CDKN2A for GBC02 with deletions and amplifications encompassing CDKN2A, FAT1 and CCND1, PIK3CA, SOX2, EGFR, MYC genes, respectively. GBC035 harbored mutations in FAT1, NOTCH1, HRAS, CDKN2A, HLA-B, HLA-A genes. While GBC035 cells showed higher E-Cadherin positive cell-cell junctions and collective cell migration in chemotaxis; GBC02 cells were vimentin-positive and demonstrated individual cell migration. Further, exhibiting their relevance to preclinical research, GBC02 3D-spheroids demonstrated enrichment of development-related gene-signatures in microarray transcriptome analysis and were resistant to Cisplatin, but showed sensitivity to cancer stem cells-targeting drug, Salinomycin. Additionally, tumorigenic ability of GBC02 was demonstrated. CONCLUSIONS: Altogether, we present here comprehensively characterized unique cell lines established from non-tobacco associated tumors, which may serve as models for preclinical investigations of oral cancers caused independent of tobacco usage.

A subtype of cancer-associated fibroblasts with lower expression of alpha-smooth muscle actin suppresses stemness through BMP4 in oral carcinoma.

Cancer-associated fibroblasts (CAFs) demonstrate the characteristics of myofibroblast differentiation by often expressing the ultrastructure of alpha-smooth muscle actin (αSMA). However, heterogeneity among cancer-associated fibroblasts (CAFs), with respect to αSMA expression, has been demonstrated in several clinical studies of oral cancer. Like normal stem cells, stem-like cancer cells (SLCCs) are also regulated extrinsically by its microenvironment; therefore, we postulated that the heterogeneous oral-CAFs would differently regulate oral-SLCCs. Using transcriptomics, we clearly demonstrated that the gene expression differences between oral tumor-derived CAFs were indeed the molecular basis of heterogeneity. This also grouped these CAFs in two distinct clusters, which were named as C1 and C2. Interestingly, the oral-CAFs belonging to C1 or C2 clusters showed low or high αSMA-score, respectively. Our data with tumor tissues and in vitro co-culture experiments interestingly demonstrated a negative correlation between αSMA-score and cell proliferation, whereas, the frequency of oral-SLCCs was significantly positively correlated with αSMA-score. The oral-CAF-subtype with lower score for αSMA (C1-type CAFs) was more supportive for cell proliferation but suppressive for the self-renewal growth of oral-SLCCs. Further, we found the determining role of BMP4 in C1-type CAFs-mediated suppression of self-renewal of oral-SLCCs. Overall, we have discovered an unexplored interaction between CAFs with lower-αSMA expression and SLCCs in oral tumors and provided the first evidence about the involvement of CAF-expressed BMP4 in regulation of self-renewal of oral-SLCCs.