Activation of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer by expanding the cancer stem cell population.

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, targets cancer stem cells and impedes resistance to antiangiogenic therapy in mouse models of breast cancer.

Antiangiogenic therapies inhibit the development of new tumor blood vessels, thereby blocking tumor growth. Despite the advances in developing antiangiogenic agents, clinical data indicate that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenics, which is attributed in part to the induction of intra-tumoral hypoxia and stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. Here, we tested whether inhibiting HIF-1α can reverse the stimulatory effects of antiangiogenic-induced hypoxia on breast CSCs. Breast cancer cells grown under hypoxic conditions were treated with the dual topoisomerase-1 (TOPO-1) and HIF-1α inhibitor camptothecin and assessed for their CSC content. In a preclinical model of breast cancer, treatment with bevacizumab was compared to the combination treatment of bevacizumab with CRLX101, an investigational nanoparticle-drug conjugate with a camptothecin payload or CRLX101 monotherapy. While exposure to hypoxia increased the number of breast CSCs, treatment with CPT blocked this effect. In preclinical mouse models, concurrent administration of CRLX101 impeded the induction of both HIF-1α and CSCs in breast tumors induced by bevacizumab treatment. Greater tumor regression and delayed tumor recurrence were observed with the combination of these agents compared to bevacizumab alone. Tumor reimplantation experiments demonstrated that the combination therapy effectively targets the CSC populations. The results from these studies support the combined administration of dual TOPO-1- and HIF-1α-targeted agents like CRLX101 with antiangiogenic agents to increase the efficacy of these treatments.

Antiangiogenic agents increase breast cancer stem cells via the generation of tumor hypoxia.

Antiangiogenic therapy has been thought to hold significant potential for the treatment of cancer. However, the efficacy of such treatments, especially in breast cancer patients, has been called into question, as recent clinical trials reveal only limited effectiveness of antiangiogenic agents in prolonging patient survival. New research using preclinical models further suggests that antiangiogenic agents actually increase invasive and metastatic properties of breast cancer cells. We demonstrate that by generating intratumoral hypoxia in human breast cancer xenografts, the antiangiogenic agents sunitinib and bevacizumab increase the population of cancer stem cells. In vitro studies revealed that hypoxia-driven stem/progenitor cell enrichment is primarily mediated by hypoxia-inducible factor 1α. We further show that the Akt/ß-catenin cancer stem cell regulatory pathway is activated in breast cancer cells under hypoxic conditions in vitro and in sunitinib-treated mouse xenografts. These studies demonstrate that hypoxia-driven cancer stem cell stimulation limits the effectiveness of antiangiogenic agents, and suggest that to improve patient outcome, these agents might have to be combined with cancer stem cell-targeting drugs.

MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells.

MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In "claudin(low)" SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFß signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated "luminal" phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

Role of microRNAs in the regulation of breast cancer stem cells.

There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by cancer stem cells (CSCs) which mediate tumor metastasis and contribute to treatment resistance and relapse. Our group was the first to describe "breast cancer stem cells" (BCSCs) characterized by expression of the cell surface markers ESA and CD44 and the absence of expression of the marker CD24. More recently, we have demonstrated that breast cancer cells contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. Interestingly, these markers identify overlapping, but not identical cell populations. Recent studies have suggested similarities between cancer stem cells and the epithelial mesenchymal transition (EMT) state. Our studies suggest that both normal and malignant breast stem cells exist in distinct, inter-convertible states (EMT and MET), the inter-conversion of which is regulated by microRNAs. EMT-like CSCs have a mesenchymal morphology, are largely quiescent, invasive and characterized by expression of the CSC markers CD24(-)CD44(+) and are EpCAM(-)CD49f(+). In contrast, the MET (mesenchymal epithelial transition) state of CSCs is characterized by active self-renewal and expression of the CSC markers ALDH and EpCAM(+)CD49f(+). A subpopulation of cells expressing both CD24(-)CD44(+) and ALDH may represent cells in transition between these states. This transition is regulated by signals originating in the microenvironment which in turn modulate microRNA networks in the CSC populations. The existence of multiple stem cell states suggests the necessity of developing therapeutic strategies capable of effectively targeting CSCs in all of these states. In addition, since CSC states are regulated by miRNAs, these small non-coding RNAs may be useful therapeutic agents to target CSCs.

Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling.

Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

A crucial role for host APCs in the induction of donor CD4+CD25+ regulatory T cell-mediated suppression of experimental graft-versus-host disease.

Allogeneic bone marrow transplantation is an effective treatment for a number of malignant and nonmalignant diseases (Applebaum. 2001. Nature. 411: 385-389 and Copelan. 2006. N Engl J Med. 354: 1813-1826). However, the application of this therapeutic modality has been impeded by a number of confounding side effects, the most frequent and severe of which is the development of graft-versus-host disease (GVHD) (Copelan. 2006. N Engl J Med. 354: 1813-1826 and Blazar and Murphy. 2005. Philos Trans R Soc Lond B Biol Sci. 360: 1747-1767). Alloreactive donor T cells are critical for causing GVHD (Fowler. 2006. Crit Rev Oncol Hematol. 57: 225-244 and Ferrara and Reddy. 2006. Semin Hematol. 43: 3-10), whereas recent data demonstrated a significant role for the naturally occurring thymic-derived donor CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) (Bluestone and Abbas. 2003. Nat Rev Immunol. 3: 253-257 and Shevach. 2006. Immunity. 25: 195-201) in suppressing experimental GVHD after bone marrow transplantation (Blazar and Taylor. 2005. Biol Blood Marrow Transpl. 11: 46-49 and Joffe and van Meerwijk. 2006. Semin Immunol. 18: 128-135) . Host APCs are required for induction of GVHD by the conventional donor T cells. However, it is not known whether they are also obligatory for donor Treg-mediated suppression of GVHD. Using multiple clinically relevant MHC-matched and -mismatched murine models of GVHD, we investigated the role of host APCs in the suppression of GVHD by donor Tregs. We found that alloantigen expression by the host APCs is necessary and sufficient for induction of GVHD protection by donor Tregs. This requirement was independent of their effect on the maintenance of Treg numbers and the production of IL-10 or IDO by the host APCs.

Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase-dependent DC functions and regulates experimental graft-versus-host disease in mice.

Histone deacetylase (HDAC) inhibitors are antitumor agents that also have antiinflammatory properties. However, the mechanisms of their immunomodulatory functions are not known. We investigated the mechanisms of action of 2 HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357, on mouse DC responses. Pretreatment of DCs with HDAC inhibitors significantly reduced TLR-induced secretion of proinflammatory cytokines, suppressed the expression of CD40 and CD80, and reduced the in vitro and in vivo allostimulatory responses induced by the DCs. In addition, injection of DCs treated ex vivo with HDAC inhibitors reduced experimental graft-versus-host disease (GVHD) in a murine allogeneic BM transplantation model. Exposure of DCs to HDAC inhibitors increased expression of indoleamine 2,3-dioxygenase (IDO), a suppressor of DC function. Blockade of IDO in WT DCs with siRNA and with DCs from IDO-deficient animals caused substantial reversal of HDAC inhibition-induced in vitro suppression of DC-stimulated responses. Direct injection of HDAC inhibitors early after allogeneic BM transplantation to chimeric animals whose BM-derived cells lacked IDO failed to protect from GVHD, demonstrating an in vivo functional role for IDO. Together, these data show that HDAC inhibitors regulate multiple DC functions through the induction of IDO and suggest that they may represent a novel class of agents to treat immune-mediated diseases.

A biomarker panel for acute graft-versus-host disease.

No validated biomarkers exist for acute graft-versus-host disease (GVHD). We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 patients who underwent transplantation that revealed 8 potential biomarkers for diagnostic of GVHD. We then measured by enzyme-linked immunosorbent assay (ELISA) the levels of these biomarkers in samples from 424 patients who underwent transplantation randomly divided into training (n = 282) and validation (n = 142) sets. Logistic regression analysis of these 8 proteins determined a composite biomarker panel of 4 proteins (interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups in the training set was 0.91 (95% confidence interval, 0.87-0.94) and 0.86 (95% confidence interval, 0.79-0.92) in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel predicted survival independently of GVHD severity. A panel of 4 biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Secondary lymphoid organs contribute to, but are not required for the induction of graft-versus-host responses following allogeneic bone marrow transplantation: a shifting paradigm for T cell allo-activation.

Graft-versus-host disease (GVHD) remains the major complication of allogeneic bone marrow transplantation (allo-BMT). GVHD fundamentally depends upon the activation of donor T cells by host antigen-presenting cells (APCs), but the precise location of these interactions remains uncertain. We examined the role of secondary lymphoid organs (SLO) in the induction of GVHD by using homozygous aly/aly mice that are deficient in lymph nodes (LNs) and Peyer's patches (PPs). Lethally irradiated, splenectomized, aly/aly (LN/PP/Sp-/-) mice and sham-splenectomized, aly/+ (LN/PP/Sp+/+) mice received BMT from either syngeneic (aly/aly) or allogeneic, major histocompatibility complex (MHC) disparate donors. Surprisingly, although LN/PP/Sp-/- allo-BMT recipients experience a survival advantage, they developed significant systemic and target organ GVHD that is comparable to LN/PP/Sp+/+ controls. Early after allo-BMT, the activation and proliferation of donor T cells was significantly greater in the BM cavity of LN/PP/Sp-/- mice compared to LN/PP/Sp+/+ controls. Donor T cells in LN/PP/Sp-/- mice demonstrated cytolytic activity in vitro, but Graft vs Leukemia (GVL) activity could be overcome by increasing the tumor burden. These data suggest that SLO contribute to, but are not required for, allogeneic T cell responses, and suggest that the BM may represent an alternative, albeit less efficient site for T cell activation following allo-BMT.

Extracorporeal photopheresis reverses experimental graft-versus-host disease through regulatory T cells.

Extracorporeal photopheresis (ECP), a technique that exposes isolated white blood cells to photoactivatable 8-methoxypsoralen and ultraviolet A radiation, is used clinically to treat cutaneous T-cell lymphoma and immune-mediated diseases such as graft-versus-host disease (GVHD). ECP is thought to control these diseases in part through direct induction of lymphocyte apoptosis, but its effects on the immune system beyond apoptosis remain poorly characterized. We have developed a novel method for incorporating ECP treatment into well-established and clinically relevant murine models of GVHD to examine its effects during an ongoing immune response. We demonstrate that the transfer of cells treated with ECP reverses established GVHD by increasing donor regulatory T cells and indirectly reducing the number of donor effector lymphocytes that themselves had never been exposed to psoralen and ultraviolet A radiation.

A role for TNF receptor type II in leukocyte infiltration into the lung during experimental idiopathic pneumonia syndrome.

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication following allogeneic stem cell transplantation (allo-SCT). Experimental models have revealed that TNF-alpha contributes to pulmonary vascular endothelial cell (EC) apoptosis, and modulates the infiltration of donor leukocytes into the lung parenchyma. The inflammatory effects of TNF-alpha are mediated by signaling through the type I (TNFRI) or type II (TNFRII) TNF receptors. We investigated the relative contribution of TNFRI and TNFRII to leukocyte infiltration into the lung following allo-SCT by using established murine models. Wild-type (wt) B6 mice or B6 animals deficient in either TNFRI or TNFRII were lethally irradiated and received SCT from allogeneic (LP/J) or syngeneic (B6) donors. At week 5 following SCT, the severity of IPS was significantly reduced in TNFRII-/- recipients compared to wt controls, but no effect was observed in TNFRI-/- animals. Bronchoalveolar lavage fluid (BALF) levels of RANTES and pulmonary ICAM-1 expression in TNFRII-/- recipients were also reduced, and correlated with a reduction of CD8(+) cells in the lung. Pulmonary inflammation was also decreased in TNFRII-/- mice using an isolated MHC class I disparate model (bm1 --> B6), and in bm1 wt mice transplanted with B6 TNF-alpha-/- donor cells. Collectively, these data demonstrate a role for TNF-alpha signaling through TNFRII in leukocyte infiltration into the lung following allo-SCT, and suggest that disruption of the TNF-alpha:TNFRII pathway may be an effective tool to prevent or treat IPS.

Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies.

BACKGROUND: The mouse is an important and widely utilized animal model for bone marrow transplant (BMT) translational studies. Here, we document the course of an unexpected increase in mortality of congenic mice that underwent BMT. METHODS: Thirty five BMTs were analyzed for survival differences utilizing the Log Rank test. Affected animals were evaluated by physical examination, necropsy, histopathology, serology for antibodies to infectious disease, and bacterial cultures. RESULTS: Severe bacteremia was identified as the main cause of death. Gastrointestinal (GI) damage was observed in histopathology. The bacteremia was most likely caused by the translocation of bacteria from the GI tract and immunosuppression caused by the myeloablative irradiation. Variability in groups of animals affected was caused by increased levels of gamma and X-ray radiation and the differing sensitivity of the two nearly genetically identical mouse strains used in the studies. CONCLUSION: Our retrospective analysis of thirty five murine BMTs performed in three different laboratories, identified C57BL/6NCr (Ly5.1) as being more radiation sensitive than B6.Cg-Ptprca/NCr (Ly5.2). This is the first report documenting a measurable difference in radiation sensitivity and its effects between an inbred strain of mice and its congenic counterpart eventually succumbing to sepsis after BMT.

RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer.

INTRODUCTION: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)-deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display "cancer stem cell" properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. EXPERIMENTAL DESIGN: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. RESULTS: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1-wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. CONCLUSIONS: Our data suggest that in both BRCA1-mutant and BRCA1-wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514-22. ©2016 AACR.

Elimination of epithelial-like and mesenchymal-like breast cancer stem cells to inhibit metastasis following nanoparticle-mediated photothermal therapy.

Increasing evidence suggesting breast cancer stem cells (BCSCs) drive metastasis and evade traditional therapies underscores a critical need to exploit the untapped potential of nanotechnology to develop innovative therapies that will significantly improve patient survival. Photothermal therapy (PTT) to induce localized hyperthermia is one of few nanoparticle-based treatments to enter clinical trials in human cancer patients, and has recently gained attention for its ability to induce a systemic immune response targeting distal cancer cells in mouse models. Here, we first conduct classic cancer stem cell (CSC) assays, both in vitro and in immune-compromised mice, to demonstrate that PTT mediated by highly crystallized iron oxide nanoparticles effectively eliminates BCSCs in translational models of triple negative breast cancer. PTT in vitro preferentially targets epithelial-like ALDH + BCSCs, followed by mesenchymal-like CD44+/CD24- BCSCs, compared to bulk cancer cells. PTT inhibits BCSC self-renewal through reduction of mammosphere formation in primary and secondary generations. Secondary implantation in NOD/SCID mice reveals the ability of PTT to impede BCSC-driven tumor formation. Next, we explore the translational potential of PTT using metastatic and immune-competent mouse models. PTT to inhibit BCSCs significantly reduces metastasis to the lung and lymph nodes. In immune-competent BALB/c mice, PTT effectively eliminates ALDH + BCSCs. These results suggest the feasibility of incorporating PTT into standard clinical treatments such as surgery to enhance BCSC destruction and inhibit metastasis, and the potential of such combination therapy to improve long-term survival in patients with metastatic breast cancer.

A Novel IL6 Antibody Sensitizes Multiple Tumor Types to Chemotherapy Including Trastuzumab-Resistant Tumors.

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance.

Lacrimal gland involvement in graft-versus-host disease: a murine model.

PURPOSE: To describe lacrimal gland involvement in a murine model of acute graft-versus-host disease (GVHD). METHODS: Histopathologic examination was performed on lacrimal glands of mice affected by GVHD at 1, 2, 4, and 6 weeks after allogeneic bone marrow transplantation (BMT). Histopathologic scoring, based on characteristic GVHD findings in human disease involved evaluation of periductal inflammation, apoptosis, ductal stasis, ductal debris, and fibrosis. CD3, CD4, CD8, CD20, and CD68 antibodies were used to stain leukocyte subsets in GVHD lacrimal gland infiltrates. Lacrimal glands from syngeneic BMT mice were used in control experiments. RESULTS: Patchy periductal inflammation and focal fibrosis were significantly elevated as early as 2 weeks after allogeneic BMT. Histopathologic scoring of lacrimal glands after allogeneic BMT was significantly different at 4 (P = 0.005) and 6 (P < 0.0001) weeks when compared with scores in syngeneic control mice. The leukocytes in lacrimal gland GVHD infiltrates were predominately CD3+ T lymphocytes, most of which were CD8+, with fewer CD4+ cells present. CONCLUSIONS: This study describes the first murine model of lacrimal gland GVHD with features that closely mimic those described in human disease and indicates that lacrimal involvement occurs in acute GVHD.

Role of CXCR3-induced donor T-cell migration in acute GVHD.

OBJECTIVE: The chemokine receptor CXCR3 has an important role in the migration of effector T cells. To investigate the role of CXCR3 on donor cells in acute graft vs host disease (GVHD) we used a well-defined experimental bone marrow transplantation (BMT) model where acute GVHD is mediated by donor CD8(+) T cells against minor histocompatibility antigens. METHODS; Lethally irradiated C3H.SW recipients were transplanted from either wild-type B6 or CXCR3(-/-) B6 donors. Donor T-cell expansion was analyzed in the spleen and small intestine of recipients by FACS. Donor T-cell function was analyzed by cytokine secretion. The severity of acute GVHD was assessed by histopathological analysis of intestine and liver, GVHD clinical scores, and survival after BMT. RESULTS: Significantly higher numbers of donor CD8(+) CXCR3(-/-) T cells were found in the spleen on days +7 and +14 compared to donor wild-type T cells. By contrast, the number of CD8(+) T cells in the small bowel of BMT recipients from CXCR3(-/-) donors was sevenfold lower than from wild-type donors. Systemic concentrations of INF-gamma and TNF-alpha were equivalent between groups. Animals that received CXCR3(-/-) donor T cells demonstrated diminished GI tract and liver damage and showed improved survival after BMT compared to recipients of wild-type donor cells (43% vs 0%, p<0.001). CONCLUSION: The migration of donor CD8(+) T cells to GVHD target organs such as the intestine depends on the expression of CXCR3 and contributes significantly to GVHD damage and overall mortality.

Breast cancer stem cells: current advances and clinical implications.

There is substantial evidence that many cancers, including breast cancer, are driven by a population of cells that display stem cell properties. These cells, termed cancer stem cells (CSCs) or tumor initiating cells, not only drive tumor initiation and growth but also mediate tumor metastasis and therapeutic resistance. In this chapter, we summarize current advances in CSC research with a major focus on breast CSCs (BCSCs). We review the prevailing methods to isolate and characterize BCSCs and recent evidence documenting their cellular origins and phenotypic plasticity that enables them to transition between mesenchymal and epithelial-like states. We describe in vitro and clinical evidence that these cells mediate metastasis and treatment resistance in breast cancer, the development of novel strategies to isolate circulating tumor cells (CTCs) that contain CSCs and the use of patient-derived xenograft (PDX) models in preclinical breast cancer research. Lastly, we highlight several signaling pathways that regulate BCSC self-renewal and describe clinical implications of targeting these cells for breast cancer treatment. The development of strategies to effectively target BCSCs has the potential to significantly improve the outcomes for patients with breast cancer.