Prevention of Inflammation-Driven Colon Carcinogenesis in Human MUC1 Transgenic Mice by Vaccination with MUC1 DNA and Dendritic Cells.

The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.

Multipeptide stimulated PBMCs generate TEM/TCM for adoptive cell therapy in multiple myeloma.

Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4+ (CD4+) and CD8+ T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naïve T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4+ and CD8+ effector and central memory T (TEM and TCM, respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ+, T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials.

Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide.

A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

BACKGROUND: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. METHODS AND RESULTS: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. CONCLUSION: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.

Chemotherapy can induce weight normalization of morbidly obese mice despite undiminished ingestion of high fat diet.

Morbidly obese patients who accomplish substantial weight loss often display a long-term decline in their resting metabolism, causing even relatively restrained caloric intake to trigger a relapse to the obese state. Paradoxically, we observed that morbidly obese mice receiving chemotherapy for cancer experienced spontaneous weight reduction despite unabated ingestion of their high fat diet (HFD). This response to chemotherapy could also be achieved in morbidly obese mice without cancer. Optimally dosed methotrexate (MTX) or cyclophosphamide (CY) enabled the mice to completely and safely normalize their body weight despite continued consumption of obesogenic quantities of HFD. Weight reduction was not attributable to decreased HFD intake, enhanced energy expenditure or malabsorption. MTX or CY dosing significantly depleted both adipose tissue and preadipocyte progenitors. Remarkably, however, despite continued high fat feeding, a compensatory increase in hepatocyte lipid storage was not observed, but rather the opposite. Gene microarray liver analyses demonstrated that HFD mice receiving MTX or CY experienced significantly inhibited lipogenesis and lipid storage, whereas Enho (energy homeostasis) gene expression was significantly upregulated. Further metabolic studies employing a human hepatocellular line revealed that MTX treatment preserved robust oxidative phosphorylation, but also promoted mitochondrial uncoupling with a surge in proton leak. This is the first report that certain optimally dosed chemotherapeutic agents can induce weight loss in morbidly obese mice without reduced dietary intake, apparently by depleting stores of adipocytes and their progenitors, curtailment of lipogenesis, and inconspicuous disposal of incoming dietary lipid via a steady state partial uncoupling of mitochondrial oxidative phosphorylation.

Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Effective adoptive immunotherapy has proved elusive for many types of human cancer, often due to difficulties achieving robust expansion of natural tumor-specific T-cells from peripheral blood. We hypothesized that antigen-driven T-cell expansion might best be triggered in vitro by acute activation of innate immunity to mimic a life-threatening infection. Unfractionated peripheral blood mononuclear cells (PBMC) were subjected to a two-step culture, first synchronizing their exposure to exogenous antigens with aggressive surrogate activation of innate immunity, followed by γ-chain cytokine-modulated T-cell hyperexpansion. Step 1 exposure to GM-CSF plus paired Toll-like receptor agonists (resiquimod and LPS), stimulated abundant IL-12 and IL-23 secretion, as well as upregulated co-stimulatory molecules and CD11c expression within the myeloid (CD33+) subpopulation. Added synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65), were reliably presented to CD4+ T-cells and cross-presented to CD8+ T-cells. Both presentation and cross-presentation demonstrated proteasomal and Sec61 dependence that could bypass the endoplasmic reticulum. Step 2 exposure to exogenous IL-7 or IL-7+IL-2 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells with a predominant interferon-γ-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other γ-chain cytokines and/or combinations were initially proliferogenic, but followed by a contractile phase not observed with IL-7 or IL-7+IL-2. Regulatory T-cells were minimally propagated under these culture conditions. This mechanistically rational culture sequence, effective even for unvaccinated donors, enables rapid preparation of T-cells recognizing tumor-associated antigens expressed by the majority of human cancers, including pancreatic cancers, breast cancers and glioblastomas.

Aberrant Glycosylation of Anchor-Optimized MUC1 Peptides Can Enhance Antigen Binding Affinity and Reverse Tolerance to Cytotoxic T Lymphocytes.

Cancer vaccines have often failed to live up to their promise, although recent results with checkpoint inhibitors are reviving hopes that they will soon fulfill their promise. Although mutation-specific vaccines are under development, there is still high interest in an off-the-shelf vaccine to a ubiquitous antigen, such as MUC1, which is aberrantly expressed on most solid and many hematological tumors, including more than 90% of breast carcinomas. Clinical trials for MUC1 have shown variable success, likely because of immunological tolerance to a self-antigen and to poor immunogenicity of tandem repeat peptides. We hypothesized that MUC1 peptides could be optimized, relying on heteroclitic optimizations of potential anchor amino acids with and without tumor-specific glycosylation of the peptides. We have identified novel MUC1 class I peptides that bind to HLA-A*0201 molecules with significantly higher affinity and function than the native MUC1 peptides. These peptides elicited CTLs from normal donors, as well as breast cancer patients, which were highly effective in killing MUC1-expressing MCF-7 breast cancer cells. Each peptide elicited lytic responses in greater than 6/8 of normal individuals and 3/3 breast cancer patients. The CTLs generated against the glycosylated-anchor modified peptides cross reacted with the native MUC1 peptide, STAPPVHNV, suggesting these analog peptides may offer substantial improvement in the design of epitope-based vaccines.

Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists.

Many cancers both evoke and subvert endogenous anti-tumor immunity. However, immunosuppression can be therapeutically reversed in subsets of cancer patients by treatments such as checkpoint inhibitors or Toll-like receptor agonists (TLRa). Moreover, chemotherapy can leukodeplete immunosuppressive host elements, including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). We hypothesized that chemotherapy-induced leukodepletion could be immunopotentiated by co-administering TLRa to emulate a life-threatening infection. Combining CpG (ODN 1826) or CpG+poly(I:C) with cyclophosphamide (CY) resulted in uniquely well-tolerated therapeutic synergy, permanently eradicating advanced mouse tumors including 4T1 (breast), Panc02 (pancreas) and CT26 (colorectal). Definitive treatment required endogenous CD8+ and CD4+ IFNγ-producing T-cells. Tumor-specific IFNγ-producing T-cells persisted during CY-induced leukopenia, whereas Tregs were progressively eliminated, especially intratumorally. Spleen-associated MDSCs were cyclically depleted by CY+TLRa treatment, with residual monocytic MDSCs requiring only continued exposure to CpG or CpG+IFNγ to effectively attack malignant cells while sparing non-transformed cells. Such tumor destruction occurred despite upregulated tumor expression of Programmed Death Ligand-1, but could be blocked by clodronate-loaded liposomes to deplete phagocytic cells or by nitric oxide synthase inhibitors. CY+TLRa also induced tumoricidal myeloid cells in naive mice, indicating that CY+TLRa's immunomodulatory impacts occurred in the complete absence of tumor-bearing, and that tumor-induced MDSCs were not an essential source of tumoricidal myeloid precursors. Repetitive CY+TLRa can therefore modulate endogenous immunity to eradicate advanced tumors without vaccinations or adoptive T-cell therapy. Human blood monocytes could be rendered similarly tumoricidal during in vitro activation with TLRa+IFNγ, underscoring the potential therapeutic relevance of these mouse tumor studies to cancer patients.

MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.

Muc1 enhances the ß-catenin protective pathway during ischemia-reperfusion injury.

The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.

Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide.

A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T-cells primed by such a vaccine candidate could be restimulated by tumor-associated MUC1.

Muc1 is protective during kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

Inhibition of adaptive immunity by IL9 can be disrupted to achieve rapid T-cell sensitization and rejection of progressive tumor challenges.

The tolerogenic cytokine IL9 promotes T regulatory cell function and allergic airway inflammation, but it has not been extensively studied in cancer. In this report, we used IL9-deficient mice to investigate the effects of IL9 in multiple models of breast and colon cancer development. Eliminating endogenous IL9 enabled sensitization of host T cells to tumors, leading to their early rejection without the requirement of vaccines or immunomodulatory therapies. Notably, IL9-deficient mice acquired immunologic memory, which actively protected from residual disease and tumor rechallenge, an effect linked to activation of CD8(+) T cells. Depletion of either CD8(+) or CD4(+) T cells abolished the benefits of IL9 loss to tumor control. Adoptive transfer experiments showed that T cells from tumor-rejecting IL9-deficient mice retained their effector competency in wild-type animals. Moreover, neutralizing IL9 antibody phenocopied the effects of IL9 gene deletion by slowing tumor progression in wild-type animals. Our results show the ability of IL9 to function as an inhibitor of adaptive immunity that prevents the formation of immunologic memory to a growing tumor, highlighting the potential for IL9 neutralization as a unique tool for cancer immunotherapy.

Immune and anticancer responses elicited by fully synthetic aberrantly glycosylated MUC1 tripartite vaccines modified by a TLR2 or TLR9 agonist.

The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.

Modification of the tumor microenvironment as a novel target of renal cell carcinoma therapeutics.

To move forward with immunotherapy, it is important to understand how the tumor microenvironment generates systemic immunosuppression in patients with renal cell carcinoma (RCC) as well as in patients with other types of solid tumors. Even though antigen discovery in RCC has lagged behind melanoma, recent clinical trials have finally authenticated that RCC is susceptible to vaccine-based therapy. Furthermore, judicious coadministration of cytokines and chemotherapy can potentiate therapeutic responses to vaccine in RCC and prolong survival, as has already proved possible for melanoma. Although high-dose interleukin 2 immunotherapy has been superseded as first-line therapy for RCC by promiscuous receptor tyrosine kinase inhibitors (rTKIs) such as sunitinib, sunitinib itself is a potent immunoadjunct in animal tumor models. A reasonable therapeutic goal is to unite antiangiogenic strategies with immunotherapy as first-line therapy for RCC. This strategy is equally appropriate for testing in all solid tumors in which the microenvironment generates immunosuppression. A common element of RCC and pancreatic, colon, breast, and other solid tumors is large numbers of circulating myeloid-derived suppressor cells (MDSCs), and because MDSCs elicit regulatory T cells rather than vice versa, gaining control over MDSCs is an important initial step in any immunotherapy. Although rTKIs like sunitinib have a remarkable capacity to deplete MDSCs and restore normal T-cell function in peripheral body compartments such as the bloodstream and the spleen, such rTKIs are effective only against MDSCs, which are engaged in phospho-STAT3-dependent programming (pSTAT3+). Unfortunately, rTKI-resistant pSTAT3- MDSCs are especially apt to arise within the tumor microenvironment itself, necessitating strategies that do not rely exclusively on STAT3 disruption. The most utilitarian strategy to gain control of both pSTAT3+ and pSTAT3- MDSCs may be to exploit the natural differentiation pathway, which permits MDSCs to mature into tumoricidal macrophages (TM1) via such stimuli as Toll-like receptor agonists, interferon γ, and CD40 ligation. Overall, this review highlights the mechanisms of immune suppression used by the different regulatory cell types operative in RCC as well as other tumors. It also describes the different therapeutic strategies to overcome the suppressive nature of the tumor microenvironment.

Downregulation of hematopoietic MUC1 during experimental colitis increases tumor-promoting myeloid-derived suppressor cells.

PURPOSE: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. EXPERIMENTAL DESIGN: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). RESULTS: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. CONCLUSION: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers.

Targeted imaging of breast tumor progression and therapeutic response in a human uMUC-1 expressing transgenic mouse model.

The ability to monitor breast cancer initiation and progression on the molecular level would provide an effective tool for early diagnosis and therapy. In the present study, we focused on the underglycosylated MUC-1 tumor antigen (uMUC-1), which is directly linked to tumor progression from pre-malignancy to advanced malignancy in breast cancer and has been identified as the independent predictor of local recurrence and tumor response to chemotherapy. We investigated whether changes in uMUC-1 expression during tumor development and therapeutic intervention could be monitored non-invasively using molecular imaging approach with the uMUC-1-specific contrast agent (MN-EPPT) detectable by magnetic resonance and fluorescence optical imaging. This was done in mice that express human uMUC-1 tumor antigen (MMT mice) and develop spontaneous mammary carcinoma in a stage-wise fashion. After the injection of MN-EPPT there was a significant reduction in average T2 relaxation times of the mammary fat pad between pre-malignancy and cancer. In addition, T2 relaxation times were already altered at pre-malignant state in these mice compared to non-tumor bearing mice. This indicated that targeting uMUC-1 could be useful for detecting pre-malignant transformation in the mammary fat pad. We also probed changes in uMUC-1 expression with MN-EPPT during therapy with doxorubicin (Dox). We observed that tumor delta-T2s were significantly reduced by treatment with Dox indicating lower accumulation of MN-EPPT. This correlated with a lower level of MUC-1 expression in the Dox-treated tumors, as confirmed by immunoblotting. Our study could provide a very sensitive molecular imaging approach for monitoring tumor progression and therapeutic response.

Myeloid-derived suppressor cells adhere to physiologic STAT3- vs STAT5-dependent hematopoietic programming, establishing diverse tumor-mediated mechanisms of immunologic escape.

The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib's profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF's capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production.

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine.

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.