We postulate that similar to bacteria, adult stem cells may also exhibit altruistic defense mechanism to protect their niche. Here, we provide preliminary data on the altruistic stem cell (ASC) based defense against a mouse corona virus; MHV-1 infection. In a mouse model of mesenchymal stem cell (MSC) mediated M. tuberculosis (Mtb) dormancy, MHV-1 infection in the lung exhibited 20 fold lower viral loads than the healthy control mice, suggesting the potential enhancement of an anti-MHV-1 defense by Mtb. This defense involved the in vivo expansion and reprogramming of CD271+ MSCs in the lung to ASC phenotype characterized by activation of genes involved in the HIF-2alpha stemness pathway. The conditioned media of the ASCs exhibited direct anti-viral activity in an in vitro model of MHV-1 induced toxicity to type II alveolar epithelial cells. MHV-1 infected Mtb harboring group versus MHV-1 alone groupexhibited an 8-fold (p<0.02; n=4) higher ASC reprogramming and 5-fold (p<0.001; n=3, student t test) higher anti-viral activity. However, ASCs facilitated intracellular replication and extracellular release of Mtb. Thus, our data suggest that MSCs exert an innate defense against MHV-1 by activating the ASC defense mechanism, which might be exploited by dormant Mtb to undergo reactivation. Hence, our findings may provide a novel anti-viral defense mechanism against novel corona virus SARS-Cov2, which could be further utilized to develop vaccine

We postulate that similar to bacteria, adult stem cells may also exhibit an altruistic defense mechanism to protect their niche against external threat. Here, we report mesenchymal stem cell (MSC) based altruistic defense against a mouse model of coronavirus, murine hepatitis virus-1 (MHV-1) infection of lung. MHV-1 infection led to reprogramming of CD271+MSCs in the lung to an “enhanced stemness” phenotype that exhibits altruistic behavior as per our previous work in human embryonic stem cells. The reprogrammed MSCs exhibited transient expansion for two weeks followed by apoptosis, and expression of stemness genes The conditioned media of the reprogrammed MSCs exhibited direct anti-viral activity in an in vitro model of MHV-1 induced toxicity to type II alveolar epithelial cells by increasing their survival/proliferation and decreasing viral load. Thus, the reprogrammed MSCs can be identified as altruistic stem cells (ASCs) which exert a unique altruistic defense against MHV-1. In a mouse model of MSC mediated Mycobacterium tuberculosis ( Mtb ) dormancy, MHV-1 infection in the lung exhibited 20-fold lower viral loads than the Mtb -free control mice on the third week of viral infection, and also exhibited 6-fold increase of ASCs, thereby enhancing the altruistic defense. Notably, these ASCs exhibited intracellular replication of Mtb , and their extracellular release. Animals showed TB reactivation suggesting that d Mtb may exploit ASCs for disease reactivation..

Natural vaccination against pathogens are known to be achieved by herd-immunity i.e. infected human host provide immunity to the community by spreading the pathogen. Whether, infected human hosts transmit vesicle packed aerosols of pathogen’s antigen for natural vaccination of the community has not yet been considered. We have explored a traditional healing method of aerosol-inoculation against small pox and tuberculosis in the Sualkuchi-Hajo cultural complex of Kamarupa, an ancient Indian region known for tantra-based healing and spirituality. In the aerosol-inoculation method against TB, selected persons with TB (later identified as smear negative TB subject) are encouraged to spread good nigudah in the community by Kirtan chanting; the good Nigudah are thought to be present within bad-nigudah or invisible krimis (tiny flesh eating living being mentioned in ancient India’s medicinal text Caraka Samhita and Atharva Veda). A 15-years of contact TB investigation study, as well as laboratory study of aerosol obtained from smear negative PTB (SN-PTB) subjects led to the identification of good Nigudah as extracellular vesicles (EVs) filled with Mtb-antigen ESAT-6. We then developed a mouse model of aerosol-inoculation using SN-PTB subject derived aerosol EVs, and identified Mtb infected mesenchymal stem cells (MSCs) of the lung as the putative source of the ESAT-6+ EVs. These Mtb infected MSCs reprogram to altruistic stem cell (ASC) phenotype, which then secrete ESAT-6+ EVs to the aerosols; healthy mice receiving the aerosol develop Mtb specific herd immunity. These results expedite our ongoing work on the innate defense mechanism of ASCs against pathogen, and provide a novel mechanism of natural vaccination, where the host extracts appropriate antigens from a pathogen, and then spread it in the community via aerosols.

Mycobacterium tuberculosis (Mtb), the causative organism of pulmonary tuberculosis (PTB) now infects more than half of the world population. The efficient transmission strategy of the pathogen includes first remaining dormant inside the infected host, next undergoing reactivation to cause post-primary tuberculosis of the lungs (PPTBL) and then transmit via aerosol to the community. In this review, we are exploring recent findings on the role of bone marrow (BM) stem cell niche in Mtb dormancy and reactivation that may underlie the mechanisms of PPTBL development. We suggest that pathogen’s interaction with the stem cell niche may be relevant in potential inflammation induced PPTBL reactivation, which need significant research attention for the future development of novel preventive and therapeutic strategies for PPTBL, especially in a post COVID-19 pandemic world. Finally, we put forward potential animal models to study the stem cell basis of Mtb dormancy and reactivation.

Tumor hypoxia and oxidative stress reprograms cancer stem cells (CSCs) to a highly aggressive and inflammatory phenotypic state of tumor stemness. Previously, we characterized tumor stemness phenotype in the ATP Binding Cassette Subfamily G Member 2 (ABCG2)–positive migratory side population (SPm) fraction of CSCs exposed to extreme hypoxia followed by reoxygenation. Here, we report that post-hypoxia/reoxygenation SPm+/ABCG2+ CSCs exerts defense against pathogen invasion that involves bystander apoptosis of non-infected CSCs. In an in vitro assay of cancer cell infection by Bacillus Calmette Guerin (BCG) or mutant Mycobacterium tuberculosis (Mtb) strain 18b (Mtb-m18b), the pathogens preferentially replicated intracellular to SPm+/ABCG2+ CSCs of seven cell lines of diverse cancer types including SCC-25 oral squamous cancer cell line. The conditioned media (CM) of infected CSCs exhibited direct anti-microbial activity against Mtb and BCG, suggesting niche defense against pathogen. Importantly, the CM of infected CSCs exhibited marked in vitro bystander apoptosis toward non-infected CSCs. Moreover, the CM-treated xenograft bearing mice showed 10- to 15-fold reduction (p &lt; 0.001; n = 7) in the number of CSCs residing in the hypoxic niches. Our in vitro studies indicated that BCG-infected SPm+/ABCG2+ equivalent EPCAM+/ABCG2+ CSCs of SCC-25 cells underwent pyroptosis and released a high mobility group box protein 1 (HMGB1)/p53 death signal into the tumor microenvironment (TME). The death signal can induce a Toll-like receptor 2/4–mediated bystander apoptosis in non-infected CSCs by activating p53/MDM2 oscillation and subsequent activation of capase-3–dependent intrinsic apoptosis. Notably, SPm+/ABCG2+ but not SP cells undergoing bystander apoptosis amplified the death signal by further release of HMGB1/p53 complex into the TME. These results suggest that post-hypoxia SPm+/ABCG2+ CSCs serve a functional role as a tumor stemness defense (TSD) phenotype to protect TME against bacterial invasion. Importantly, the CM of TSD phenotype undergoing bystander apoptosis may have therapeutic uses against CSCs residing in the hypoxic niche.

Background: India's North East is an economically deprived and politically unstable region inhabited by diverse groups of indigenous people. The incidence of cancer (mainly oral) is unusually high in the region. Through our KaviKrishna telemedicine care (www.kavikrishnalab.org) with collaboration of Thoreau Lab for Global Health (www.thoreaulab.org) we have initiated a longitudinal study in the greater Sualkuchi (50,000 population) area to identify different elements that underlie cancer health disparities. Specially, we wanted to develop an indigenous knowledge system (IKS) based approach (1) as an analytical tool to study cancer health disparities. IKS may be defined as a knowledge-emergence mechanism as a result of non-linear communication between an individual and his/her culture (1). Methods: An IKS-based experimental approach was developed as follows. First, we identified 45 cancer patients (mostly oral cancer, age group- 30-65 years; 60% male) living in Sualkuchi. Second, we mapped out each patient’s social network/support system through regular home visit, interviews, focused group discussion, and visits to attending doctors. Third, one group of patients (n= 30 out of 45 patients) and relatives were empowered with basic knowledge about their disease and the available treatments needs through regular focused group meetings. After three months, we repeated interviews/group discussion to find whether they communicated their knowledge/experience to generate an IKS-based system (1). The data were then fed into the IKS-network analysis tool, which we developed partly, based on hub-system (3) and thematic network based approach (3). 10 patients from urban area served as a control population. Results: We found that 40 out of 45 patients find it extremely difficult to navigate the complex cancer care system due to lack of communication with treating oncologists/surgeons as compared to urban patients. 15 patients discontinued follow up visit mainly for this reason alone. 25 patients showed evidence of communicating through a pre-existing IKS-based system. Surprisingly, these 25 patients were positively responding to our care services/group discussion and becoming emotionally and psychologically strong towards managing their self-care. Conclusion: Our study indicates that lack of communication is a major cause of cancer health disparity. An IKS based approach may enhance communication, and contribute to cancer care in rural India. 1. Bikul Das. Globalization and Emerging Opportunities of Indigenous Culture. 2003. (www.academia.edu/ 7882695).2. Anita Kothari et al. Using an integrated knowledge translation approach to build a public health research agenda. Healthy Research Policy and Systems, 2014. (PMID: 24475759).3. Jennifer Attride-stirling: Thematic networks: an analytic tool for qualitative research. 2001. Qualitative Research. Note: This abstract was not presented at the meeting. Citation Format: Lekhika Pathak, Bidisha Pal, Tutumoni Baishya, Anupam Sarma, Bikul Das. A novel indigenous knowledge system based approach to study cancer health disparities in rural population of North East India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3342.

The mechanism of HPV16 induced malignant transformation of oral epithelial cells is not yet clear. We speculated that HPV16 may induce stem cell altruism that reprogram specific subfraction of the keratinocyte population to enhanced stemness state, a previously reported pre-malignant phenotype in human ES cells by our lab (1). Indeed, using HPV16 E6 transduction method, we demonstrated that CD271+ primary human keratinocytes undergo enhanced stemness switch i.e. low p53 activity, and high expression of HIF-2a, Nanog, sox2 and oct4. Importantly, these cells indicated resistance to calcium and serum induced differentiation, and clonogenic activities and failed to undergo malignant transformation (2). Considering that HPV16 induced carcinogenesis may involve additional steps to transformation, next, we treated CD271+ cells from HPV16 E6 transduced cells with 4-NQO oral carcinogen. We found that 4-NQO tx induced the MYC-HIF-2a stemness pathway (3), which we recently characterized in cancer stem cells (4). However, MYC activity was transient and therefore, the cells did not exhibit reprograming of enhanced stemness to tumor stemness state, the later being identified in cancer stem cells (5). Thus, it seems that additional oncogenes, in addition to MYC may be involved in the reprograming of enhanced stemness to tumor stemness state. Here we evaluate the ability of oral bacteria F. nucleatum (F.N.) to accelerate malignant process and facilitate the enhanced stemness to acquire tumor stemness state. Previously, we showed that F.N. can induce HIF-2a stemness pathway in oral cancer cell line (6), which prompted us to use this model for this present work. Considering that F.N. surface protein Fap2 can modulate E cadherins, we speculated that F.N. might induce E. cadherin-EGFR axis to promote ras oncogene activity in collaboration with HPV16. Methods: The HPV16 E6 transduced (by using the retroviral LXSN 16E6 system) primary oral keratinocytes were grown for two weeks as previously described (2-3). In these cells. F.N. was added in 1:10 MOI for 12 hours under anerobic condition (5), and then washed and cultured in vitro for 2 weeks. The CD271+ cells were then immunomagnetically sorted and subjected to evaluation for MYC/HIF2 stemness pathway. Because, ABCG2+ cells are endowed with this stemness pathway (1,5), we next sorted the CD271+/ABCG2+ cells. Results: We found that addition of F.N. led to upregulation of MYC/HIF-2a stemness pathway in the CD271+/ABCG2+ cells. These cells showed rapid proliferation for 2-3 weeks, and resistance to calcium and serum induced terminal differentiation and activated the EGFR/HRas axis. Interestingly, F.N. lysate is sufficient to induce EGFR expression and the activation of MYC/stemness pathway in these E6 transduced cells. Notably silencing of E-cadherin by siRNA led to loss of F.N mediated induction of MYC and Ras, and spontaneous apoptosis of the cells. Conclusions: These data indicate that oral bacteria like F.N. can cause enhanced stemness switch, a pre-malignant state by activating the EGFR/Ras pathway that activate MYC/stemness pathway. 1. Das B,etal. Stem Cells. 2012 2. AACR Abstract nr 4297 3. AACR Abstract nr 4073 4. Das B et al. Cancer Res. 2019 5. AACR Abstract nr 3064. Citation Format: Ibrahim Akeel, Bidisha Pal, Partha Jyoti Saikia, Lekhika Pathak, Sandhya Sorra, Bikul Das. Fusobacterium Nucleatum and HPV16 cooperate to reprogram the human primary oral keratinocyte to enhanced stemness state, a newly identified pre-malignant cell state [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3821.

Background: Our laboratory has identified tumor stemness phenotype in several tumor types including lymphoma, oral cancer, neuroblastoma, and sarcomas. In neuroblastoma, we found that stress induction including hypoxia, chemotherapy regimens, etc., activates the tumor stemness pathways including the HIF-2alpha stemness pathway. In lymphoma, we found that MYC regulates the HIF-2alpha stemness pathway to maintain tumor stemness phenotype of lymphoma CSCs (PMID:31266772). In this study, we evaluated the tumor stemness phenotype in breast cancer. Methods: MCF-7 breast cancer cell line was subjected to hypoxia/oxidative stress for three-days and further flow-sorted to obtain the highly tumorigenic ABCG2+ cells population. Next, these cells were evaluated for tumor stemness phenotype (Nanog, Sox2, Oct4, p53, MYC, and HIF-2a) via quantitative PCR. Induction of EMT (epithelial-mesenchymal transition) the phenotype was evaluated via qPCR using the known markers such as Snail, Twist, and vimentin. Invitro/ invivo self-renewal capacity of ABCG2+ cells was evaluated by clonogenic assays and serial transplantation assay. siRNA treatment was performed to transiently knockdown and observe changes in phenotype. Results: ABCG2+ cells derived from post- hypoxia treated MCF-7 indicated tumor stemness phenotype with high gene expression levels of Nanog, Sox2, Oct4, p53, MYC, and HIF-2a. Gene expression of EMT markers was markedly high in comparison to ABCG2- cell population. Hypoxia treatment amplified the self-renewal capacity in ABCG2+ cells, while the loss of HIF-2a using siRNA treatment (in-vitro) or FM19G11 treatment (potent invivo chemical HIF inhibitor) abrogated the self-renewal capacity as observed by fewer colonies and absence of tumors. Associated reduction in the stemness phenotype and EMT was observed in siRNA HIF-2a treated ABCG2+ cells. Importantly we found that in these cells, TLR-2 and Nfκβ become activated following HMGB-1 ligand binding, suggesting the pro-inflammatory phenotype of these cells. Additionally, these cells showed a high expression of the aryl hydrocarbon receptor (AhR). Conclusion: These data suggest that MCF-7, upon exposure to hypoxia/oxidative stress undergo tumor stemness switch and activate the MYC-HIF-2alhpa stemness pathway. Our study also shows that the tumor stemness phenotype in his cell line incorporates the activation of the TLR/HGMB1 axis and also the AhR pathway. We also found that loss of HIF-2a negatively affected the tumor stemness phenotype of ABCG2+ breast cancer stem cells. Thus, targeting the MYC-HIF-2alpha stemness pathway may prove to be therapeutically beneficial. Citation Format: Joyeeta Talukdar, Sorra Sandhya, Lekhika Pathak, Zoya Mann, Bidisha Pal, Seema Bhuyan, Anurag Shrivastav, Sujata Mohanty, Bikul Das. Identification of breast cancer cells exhibiting tumor stemness phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3565.