Masoud H. Manjili

Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

Therapeutic Cancer Vaccines: Past, Present and Future

Therapeutic vaccines represent a viable option for active immunotherapy of cancers that aim to treat late stage disease by using a patients own immune system. The promising results from clinical trials recently led to the approval of the first therapeutic cancer vaccine by the U.S. Food and Drug Administration. This major breakthrough not only provides a new treatment modality for cancer management but also paves the way for rationally designing and optimizing future vaccines with improved anticancer efficacy. Numerous vaccine strategies are currently being evaluated both preclinically and clinically. This review discusses therapeutic cancer vaccines from diverse platforms or targets as well as the preclinical and clinical studies employing these therapeutic vaccines. We also consider tumor-induced immune suppression that hinders the potency of therapeutic vaccines, and potential strategies to counteract these mechanisms for generating more robust and durable antitumor immune responses.

CD44+/CD24−/low cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome☆

Women classified as having triple-negative tumors have a poor prognosis. The importance of CD44(+)/CD24(-/low) (stem/progenitor cell-phenotype) in breast cancer patients has also been appreciated. However, correlation between triple negativity and CD44(+)/CD24(-/low) with tumor recurrence remains elusive. In the present study, we evaluated tumor specimens of 50 breast cancer patients with known hormone receptor status for whom we had follow-up information and outcome data available, and performed immunohistochemistry analysis to determine CD44 and CD24 expression. Gene expression arrays were also independently performed on 52 breast cancer specimens with banked frozen tissue. Lastly, we used FVBN202 transgenic mouse model of breast carcinoma and determined the hormone receptor status, the proportion of CD44(+)/CD24(-/low) breast cancer stem-like cells, and the behavior of the tumor. We determined that patients with triple-negative tumors had significantly higher incidence of recurrence or distant metastasis associated with increased frequency of breast cancer stem cell phenotypes compared with those with non-triple-negative tumors. Preclinical studies in FVBN202 transgenic mice confirmed these findings by showing that relapsed tumors were triple negative and had significantly higher frequency of breast cancer stem cells compared with their related primary tumors. Unlike non-triple-negative primary tumors, relapsed triple-negative tumors were tumorigenic at low doses when inoculated into FVBN202 transgenic mice. These findings suggest that CD44(+)/CD24(-/low) breast cancer stem-like cells play an important role in the clinical behavior of triple-negative breast cancer and that development of therapeutic targets directed to breast cancer stem-like cells may lead to reduction in the aggressiveness of triple-negative breast cancers.

GM-CSF is one of the main breast tumor-derived soluble factors involved in the differentiation of CD11b-Gr1- bone marrow progenitor cells into myeloid-derived suppressor cells

Recent reports have shown the involvement of tumor burden as well as GM-CSF in supporting myeloid-derived suppressor cells (MDSC). However, it is not known what progenitor cells may differentiate into MDSC in the presence of GM-CSF, and whether FVBN202 transgenic mouse model of spontaneous breast carcinoma may exhibit distinct subset distribution of CD11b+Gr1+ cells. In addition, it is not known why CD11b+Gr1+ cells derived from tumor-free and tumor-bearing animals exhibit different functions. In this study, we determined that GM-CSF was one of the tumor-derived soluble factors that induced differentiation of CD11b-Gr1- progenitor cells from within monocytic/granulocytic bone marrow cells into CD11b+Gr1+ cells. We also showed that CD11b+Gr1+ cells in FVBN202 mice consisted of CD11b+Ly6G-Ly6C+ suppressive and CD11b+Ly6G+Ly6C+ non-suppressive subsets. Previously reported variations between tumor-free and tumor-bearing animals in the function of their CD11b+Gr1+ cells were found to be due to the variations in the proportion of these two subsets. Therefore, increasing ratios of CD11b+Gr1+ cells derived from tumor-free animals revealed their suppressive activity on T cells, in vitro. Importantly, GM-CSF supported the generation of CD11b+Ly6G-Ly6C+ suppressor subsets that inhibited proliferation as well as anti-tumor function of neu-specific T cells. These findings suggest revisiting the use of GM-CSF for the expansion of dendritic cells, ex vivo, for cell-based immunotherapy or as an adjuvant for vaccines for patients with cancer in whom MDSC play a major role in the suppression of anti-tumor immune responses.

HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice.

Heat shock proteins (HSPs) are shown to be strong immunoadjuvants, eliciting both innate and adaptive immune responses against cancers. HSP110 is related in sequence to HSP70 and is ∼4-fold more efficient in binding to and stabilizing denatured protein substrates compared with HSP70. In the present study we evaluated the ability of a heat shock complex of HSP110 with the intracellular domain (ICD) of human HER-2/neu to elicit effective antitumor immune responses and to inhibit spontaneous mammary tumors in FVB-neu (FVBN202) transgenic mice. The HSP110-ICD complex was capable of breaking tolerance against the rat neu protein and inhibiting spontaneous mammary tumor development. This vaccine induced ICD-specific IFN-γ and IL-4 production. Depletion studies revealed that CD8+ T cells were involved in protection against challenge with mouse mammary tumors, whereas CD4+ T cells revealed partial protection. Increased IgG2a Ab titer in the sera of tumor-free animals after vaccination and elevated CD4+ CD25+ regulatory T cells in the PBL of tumor-bearing animals suggested that IFN-γ-producing Th1 cells may be responsible for partial protection of CD4+ T cells against the mammary tumor challenge, whereas CD4+CD25+ regulatory T cells (Th2 cells) may suppress the antitumor immune responses. Together, these results suggest that HSP110-ICD complex can elicit effective IFN-γ-producing T cells against spontaneous mammary tumors and that up-regulation of CD4+ CD25+ regulatory T cells may prevent complete eradication of the tumor following immunotherapy.

HER-2/neu antigen loss and relapse of mammary carcinoma are actively induced by T cell-mediated anti-tumor immune responses

Induction of tumor‐specific immune responses results in the inhibition of tumor development. However, tumors recur because of the tumor immunoediting process that facilitates development of escape mechanisms in tumors. It is not known whether tumor escape is an active process whereby anti‐tumor immune responses induce loss or downregulation of the target antigen in the antigen‐positive clones. To address this question, we used rat neu‐overexpressing mouse mammary carcinoma (MMC) and its relapsed neu antigen‐negative variant (ANV). ANV emerged from MMC under pressure from neu‐specific T cell responses in vivo. We then cloned residual neu antigen‐negative cells from MMC and residual neu antigen‐positive cells from ANV. We found marked differences between these neu‐negative clones and ANV, demonstrating that the residual neu‐negative clones are probably not the origin of ANV. Since initial rejection of MMC was associated with the presence of IFN‐γ‐secreting T cells, we treated MMC with IFN‐γ and showed that IFN‐γ could induce downregulation of neu expression in MMC. This appears to be due to methylation of the neu promoter. Together, these data suggest that neu antigen loss is an active process that occurs in primary tumors due to the neu‐targeted anti‐tumor immune responses.

Human T cells express CD25 and Foxp3 upon activation and exhibit effector/memory phenotypes without any regulatory/suppressor function

BackgroundFoxp3 has been suggested to be a standard marker for murine Tregs whereas its role as marker for human Tregs is controversial. While some reports have shown that human Foxp3+ T cells had no regulatory function others have shown their role in the inhibition of T cell proliferation.MethodsT cell activation was performed by means of brayostatin-1/ionomycin (B/I), mixed lymphocyte reaction (MLR), and CD3/CD28 activation. T cell proliferation was performed using BrdU and CFSE staining. Flow cytometry was performed to determine Foxp3 expression, cell proliferation, viabilities and phenotype analyses of T cells.ResultsBoth CD4+ and CD8+ T cells expressed Foxp3 upon activation in vitro. Expression of Foxp3 remained more stable in CD4+CD25+ T cells compared to that in CD8+CD25+ T cells. The CD4+CD25+Foxp3+ T cells expressed CD44 and CD62L, showing their effector and memory phenotypes. Both FoxP3- responder T cells and CD4+FoxP3+ T cells underwent proliferation upon CD3/CD28 activation.ConclusionExpression of Foxp3 does not necessarily convey regulatory function in human CD4+CD25+ T cells. Increased FoxP3 on CD44+ effector and CD44+CD62L+ memory T cells upon stimulation suggest the activation-induced regulation of FoxP3 expression.

An immunologic portrait of cancer

The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Here, we focus on an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells. As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process. Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Moreover, these signatures overlap with those observed during immune-mediated cancer rejection and, more broadly, immune-mediated tissue-specific destruction in other immune pathologies. Thus, a discussion concerning this cancer phenotype is warranted as it remains unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Here we reflect on commonalities and discrepancies among studies and on the genetic or somatic conditions that may cause this schism in cancer behavior.

Cutting Edge: Mast Cells Critically Augment Myeloid-Derived Suppressor Cell Activity

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (KitWsh/Wsh) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells

Adoptive immunotherapy (AIT) using ex vivo-expanded HER-2/neu-specific T cells has shown initial promising results against disseminated tumor cells in the bone marrow. However, it has failed to promote objective responses against primary tumors. We report for the first time that alternating gamma chain cytokines (IL-2, IL-7 and IL-15) ex vivo can expand the neu-specific lymphocytes that can kill breast tumors in vitro. However, the anti-tumor efficacy of these neu-specific T cells was compromised by the increased levels of myeloid-derived suppressor cells (MDSC) during the premalignant stage in FVBN202 transgenic mouse model of breast carcinoma. Combination of AIT with the depletion of MDSC, in vivo, resulted in the regression of neu positive primary tumors. Importantly, neu-specific antibody responses were restored only when AIT was combined with the depletion of MDSC. In vitro studies determined that MDSC caused inhibition of T cell proliferation in a contact-dependent manner. Together, these results suggest that combination of AIT with depletion or inhibition of MDSC could lead to the regression of mammary tumors.