Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mainly attacks the respiratory system and is characterized by pneumonia, cytokine storm, coagulation disorders and severe immune downregulation. Although public health experts predicted worst outcomes in Africa, the incidence, hospitalization and mortality rates have been lower in Africa compared to other continents. Interestingly, lower incidence and mortality rates have been observed in women from Africa compared to their cohorts from other continents. Also, in the US non-Hispanic Black females have lower COVID-19 and death rates compared to their white counterparts. It's unclear why this significant difference exists; however, the ovarian function, genetics and immunological statuses could play a major role. Women of African descent have elevated levels of estrogen compared with Caucasians hence we anticipate that estrogen might offer some protection against the SARS-CoV-2 infections. The racial differences in lifestyle, age and inaccessibility to contraceptive usage might also play a role. Here, we provide insight on how the high levels of estrogen in African women might contribute to the lower cases and fatalities in Africa. Specifically, estrogen might offer protection against COVID-19 by suppressing hyper-production of cytokines, promoting anti-inflammatory cytokines, stimulating antibody production and suppressing endoplasmic reticulum (ER) stress. This will as well provide useful information on how future pandemics could be managed using Africa as a case study.
Abstract High-grade serous carcinoma (HGSC) is a deadly malignancy leading to ~70% of the 140,000 ovarian cancer deaths each year. Resistance to platinum chemotherapy followed by recurrence and an incurable disease occurs in most HGSC patients. Contemporary immune checkpoint blockade therapies have shown minimal efficacy in this cancer. Our previous investigations established that tumour interferon (IFN) activation status and CD8+ T cell density are predictors of chemotherapy response in HGSC. Furthermore, we also showed that the IFN induced chemokine CXCL10 is a key determinant of increased survival via immune cell recruitment in the tumour immune microenvironment (TIME). Given that signal transducer and activator of transcription 1 (STAT1) is central to the feed forward loops of cellular IFN responses, we investigated STAT1 associated transcriptomic alterations and spatial profiles of immune cells in 204 pre-treatment HGSC tumours. RNA-sequencing based whole transcriptomic profiling revealed that higher STAT1 expression significantly correlated with higher immunomodulatory gene expression, including immune checkpoints and activators, in both chemotherapy sensitive and resistant tumours. Findings were independently validated in a cohort of 379 HGSC tumour RNA-Seq profiles from The Cancer Genome Atlas Network ovarian cancer dataset. Multiplex immunofluorescence based spatial profiling of CD8+ T cells, FoxP3+ T regulatory T cells, CD68+M1, and CD163+ M2 macrophages and expression of PD-L1, PD-1, IDO1 immune checkpoints was performed. Findings from our study provide evidence for IFN mediated adaptive immune resistance in the HGSC TIME and will potentially inform the design of rational chemo-immunotherapy approaches.
Abstract High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with high rates of chemotherapy resistance and poor outcomes. Our previous studies have demonstrated the variable tumor immune microenvironment states that associate with platinum chemotherapy response. We further showed the significance of the interferon (IFN) induced chemokine CXCL10 as a key mediator of tumor infiltrating immune cell recruitment. Using the ID8-Trp53−/− murine model of HGSC, we demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC tumors to carboplatin chemotherapy and sensitizing them to immune checkpoint blockade therapy through a heightened type 1 IFN (IFN1) response. CXCL10 production via IFN1 is also governed by genes that regulate cellular DNA damage repair pathways. Evolving evidence indicates a role of BRCA1 and PTEN genes in mediating cellular IFN1 responses. Losses in the function of these genes is widely prevalent in a large proportion of HGSC tumors, where tumors with BRCA1 mutations (~25% of HGSC cases) have higher CD8+ T cell infiltration in contrast to those with loss of PTEN (~10% of cases). We hypothesized that HGSC tumors with loss of PTEN expression can be rendered susceptible to immune mediated killing via activating the STING pathway. Tumors generated from ID8-Trp53−/−; Brca1−/− cells and those from ID8-Trp53−/−; Pten−/− cells in C57BL6 mice showed significant immunologic differences through local and systemic immune profiling. The addition of STING agonist treatment significantly increased chemosensitivity and improved overall response in mice implanted with ID8-Trp53−/−; Pten−/− cells compared to those treated with carboplatin alone, altering immune responses. This study is foundational to inform rational combinations of STING pathway activating therapies in HGSC, augmenting responses to existing chemotherapy regimens and prolonging survival rates in patients. Citation Format: Noor Shakfa, Elizabeth Lightbody, Deyang Li, Juliette Wilson-Sanchez, Gwenaelle Conseil, Afrakoma Afriyie-Asante, Stephen Chenard, Ali Hamade, Madhuri Koti. Improving genotype specific chemotherapy response in ovarian cancer via cGAS-STING pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1708.
Hepatocellular carcinoma (HCC) is a primary liver cancer characterised by mutations in the cellular machinery, which induces the liver cells to replicate at a higher rate. It is the fifth most common cancer and the third most common cause of cancer-related deaths worldwide. The extensive prevalence of this malignancy carries a significant economic burden on a country. HCC occurs within the background of cirrhosis and chronic liver diseases, where hepatitis B virus (HBV), hepatitis C virus (HCV) infections and alcoholism are regarded as the most frequent risk factors of the disease. However, current diagnostic tools are sensitive only at the advanced stages, which hugely contribute to the grave prognosis and compromised effective curative treatment, resulting in poor 5-year patient survival. With the continuous devastating effect of this malignancy, there is an urgent need for an efficient tool, which could detect the malignancy at both the early and late stages.
Recently, an increasing number of studies have shown that cancer patients produce detectable serological biomarkers in the form of IgG autoantibodies, which appear five years even before clinical manifestation of the disease. This evidence has recently generated opportunities for immune system exploitation as source of cancer biomarkers. Autoantibodies are elicited by the release of tumour-associated antigens (TAAs) during tumour development, making the autoantibody signature an effective approach for the early diagnosis of HCC. They could also represent as novel tools for prognosis, monitoring and prediction of response for chemotherapy. It is therefore crucial to design a non-invasive, high-throughput and robust screening system that would allow autoantibodies to be detected early in HCC and high-risk patients.
In recent times, the antigen microarray, which is a new generation probing technique, has emerged as a promising tool for the examination of humoral immune responses in cancer in a multiplexed and high-throughput manner. This technology is adapted as an alternative approach to the already existing Enzyme Linked Immunosorbent Assay (ELISA). In Antigen Microarrays, the recombinant TAAs are spotted robotically on a glass slide and probed with sera after which signals are amplified and quantified through fluorescence. The technology has the advantage of quantifying serum autoantibodies to hundreds of TAAs simultaneously and further providing a form of protein analysis at a scale beyond that which is achievable by traditional immunological techniques.
This thesis documents the development, validation and application of the in-house Antigen Microarray platform to detect and characterise specific anti-TAA IgG autoantibodies to a panel of TAAs in the sera of HCC and underlying liver disease patients.
Important parameters associated with the antigen microarray such as slide surface chemistry, target humidity, printing buffer, blocking buffer and antigen concentration were optimised to improve the accuracy and precision of the technology for this early biomarker detection. Secondly, the validity of the developed technology was further assessed using guidelines drawn by the Food and Drug Administration for pharmacokinetic assay validation. Additionally, we opted to produce a panel of eight recombinant TAAs in a mammalian expression system, to be sure of the quality and purity of antigens generated. The suitability of in-house TAAs were also tested on the microarray assay to reveal precision of the assay. Lastly, serum anti-TAA autoantibody levels were measured in HCC (n=183), liver cirrhosis (n=90), chronic hepatitis patients (n=133) as well as healthy groups (n=46), over a wide range of in-house (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) and commercial TAAs produced both in mammalian (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) and E. coli expression systems (KRAS, HRAS, HDGF, RALA, P62, APCN, AFP, IMP1, GRP78, Gankyrin).
Validation studies showed high reproducibility as precision values were within the acceptable limits, namely, intra (within) and inter (across days) coefficient of variation were less than 15% and 20% respectively. Eight highly expressed Flag-tagged recombinant TAAs (KRAS, HRAS, HDGF, CDKN2A, RALA, P62, P53, Gankyrin) in lysates were produced and evaluated by western blotting.
Preliminary studies conducted to investigate whether heat treatment reduces or elevates autoantibody levels in sera revealed no significant effect (P>0.05) of heat inactivation on the detectability of autoantibodies to TAAs. There was a significant difference (p<0.05) in the distribution of specific anti-TAA autoantibody responses across the four cohorts with highest responses detected in HCC patients followed by chronic hepatitis and liver cirrhosis patients. This suggests that high anti-TAA autoantibody levels are strongly associated with the cancer. Poor correlation (r<1) was observed between autoantibody responses and the gender or age of HCC patients. Positive autoantibody frequency to the three TAA categories in HCC patients varied from 3.0% to 23% with highest frequencies observed in responses to P62 (18%), HRAS (20%) and HDGF (21%). Elevated autoantibody prevalence to HRAS (19.5%-25%) and P53 (9.7%-10.5%) were also detected in pre-malignant liver conditions (chronic liver hepatitis) suggesting malignant transition to HCC is related with increased responses to cellular proteins which might play a role in the cancer.
Our study has explored the development of a simple blood test which has the potential to offer a competitive benefit over present HCC diagnostic tools as it is easy to use, portable, non-invasive and inexpensive. It could also be applicable as an annual check in General Practice (GP) surgery to provide reports of greater relevance that could be used for effective curative treatment strategies.
Background High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor gene loss. Given the type 1 interferon regulatory function of BRCA1 and PTEN genes and their associated contrasting T-cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, respectively, in this study we investigated the potential of stimulator of interferon genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency. Methods Expression of PTEN protein was evaluated in tissue microarrays generated using pretreatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8- Trp53 –/– ; Pten –/– and ID8- Trp53 –/– ; Brca1 –/– , were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy. Results Patient tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intraepithelial CD68+ macrophage infiltration as compared with intact PTEN expression. In vivo studies demonstrated that Pten -deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+MDSCs in the ascites, and reduced effector CD8+ cytotoxic T-cell function compared with Brca1 -deficient cells; further, tumors from mice injected with Pten -deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten -deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten -deficient ascites to M1-like phenotype and rescued CD8+ cytotoxic T-cell activation. Conclusions This study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN-deficient HGSC.
Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK+) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK+ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK+ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK+ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.
Abstract High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy, with high rates of chemotherapy resistance and poor outcomes. We previously showed that tumors from chemotherapy-resistant patients show an immunologically cold phenotype, exhibiting lower density of CD8+ T cells and low expression of interferon genes. Subsequently, using the ID8-Trp53−/− murine model of HGSC, we further demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC to carboplatin chemotherapy. Currently, we aim to better characterize the effect of cancer cell genotype on response to treatment in HGSC. The evolution of the tumor immune microenvironment (TIME) as immunologically hot or cold can be to some extent dictated by cancer cell-specific genetic alterations. While loss of the tumor suppressor TP53 is a universal mutation (>96% of cases), previous reports in HGSC have shown that patients with BRCA1 mutations (~25% of cases) have higher CD8+ T-cell infiltration and higher chemosensitivity. In contrast, loss of PTEN (seen in ~20% of cases) is associated with poor outcomes and chemoresistance. We hypothesized that HGSC tumors with loss of PTEN expression can benefit via immunomodulatory treatment approaches that activate the STING pathway following chemotherapy. C57/BL6 mice were implanted with either ID8-Trp53−/−; Brca1−/− cells or ID8-Trp53−/−; Pten−/− cells and subjected to one of three treatment groups: vehicle, carboplatin, or carboplatin + STING agonist. Characterization of the TIME generated from ID8-Trp53−/−; Brca1−/− cells, and those from ID8-Trp53−/−; Pten−/− cells, through local and systemic immune profiling, showed significant immunologic differences between different genotypes. Addition of STING agonist significantly increased chemosensitivity and improved overall response in ID8-Trp53−/−; Pten−/− injected mice compared to those treated with carboplatin alone. This study helps to determine the potential of STING pathway activation in inducing an activated tumor immune state in genotypes of HGSC that result in a “cold” TIME, such as loss of PTEN, to augment responses to chemotherapy and prolong survival. Citation Format: Noor Shakfa, Elizabeth Lightbody, Afrakoma Afriyie-Asante, Vinicius Kannen, Madhuri Koti. Improving chemotherapy response of immunologically cold high-grade serous ovarian cancer with loss of PTEN using STING agonist [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B78.
ABSTRACT Background High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by chemoresistance and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor gene loss. Loss of BRCA1 and PTEN function due to mutations or epigenetic influence have been widely associated with variable clinical outcomes, where tumors with BRCA1 mutations exhibit increased chemosensitivity and those with PTEN mutations have been reported to exhibit chemoresistance. Given the established type 1 interferon regulatory function of BRCA1 and PTEN genes and associated contrasting T cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, in this study we investigated the potential of Stimulator of Interferon Genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency. Methods Expression of PTEN protein was evaluated in tissue microarrays generated using pre-treatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8- Trp53 -/- ; Pten -/- and ID8- Trp53 -/- ; Brca1 -/- , were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy. Results Tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intra-epithelial CD68+ macrophage infiltration as compared to intact PTEN expression. In vivo studies demonstrated that Pten deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+ MDSCs in the ascites, and reduced effector CD8+ cytotoxic T cell function compared to Brca1 deficient cells; further, tumors from mice injected with Pten deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten deficient ascites to a M1-like phenotype and rescued CD8+ cytotoxic T cell activation. Conclusions This study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN deficient HGSC.
Abstract High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with high rates of chemotherapy resistance and poor outcomes. Our previous studies have demonstrated the variable tumor immune microenvironment states that associate with platinum chemotherapy response. We further showed the significance of the interferon induced chemokine CXCL10 as a key mediator of tumor infiltrating immune cell recruitment. Using the ID8-Trp53−/− murine model of HGSC, we demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC tumors to carboplatin chemotherapy and sensitizing them to immune checkpoint blockade. Notably, CXCL10 production via IFN1 is also governed by genes that regulate cellular DNA damage repair pathways. Evolving evidence indicates a role of BRCA1 and PTEN genes in mediating cellular IFN1 responses. Losses in the function of these genes is widely prevalent in a large proportion of HGSC tumors, where tumors with BRCA1 mutations have higher CD8+ T cell infiltration in contrast to those with loss of PTEN. We hypothesized that HGSC tumors with loss of PTEN expression can be rendered susceptible to immune mediated killing via activating the STING pathway. Tumors generated from ID8-Trp53−/−; Brca1−/− cells and those from ID8-Trp53−/−; Pten−/− cells in C57BL6 mice showed significant immunologic differences. STING agonist treatment significantly increased chemosensitivity and improved overall response in mice implanted with ID8-Trp53−/−; Pten−/− cells compared to those treated with carboplatin alone, altering immune responses. This study is foundational to guide rationalistic combinations of STING pathway activating therapies in HGSC.
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