J. Alejandro Lopez

Antigens for cancer immunotherapy.

Progress in tumor immunology has not been translated to effective immunotherapies for cancer. Most of the current effort in basic and clinical research concentrates on generating effective immune responses against model or well characterized antigens, yet vaccines targeting defined antigens have been less clinically successful than those based on whole tumor cells or their extracts. This review considers characteristics of proteins that determine how effectively they might serve as targets of immune control, and how different sources of antigens have fared in clinical trials.

Breast cancer stem cells: implications for therapy of breast cancer

The concept of cancer stem cells responsible for tumour origin, maintenance, and resistance to treatment has gained prominence in the field of breast cancer research. The therapeutic targeting of these cells has the potential to eliminate residual disease and may become an important component of a multimodality treatment. Recent improvements in immunotherapy targeting of tumour-associated antigens have advanced the prospect of targeting breast cancer stem cells, an approach that might lead to more meaningful clinical remissions. Here, we review the role of stem cells in the healthy breast, the role of breast cancer stem cells in disease, and the potential to target these cells.

Breast cancer stem cells: Treatment resistance and therapeutic opportunities

The clinical and pathologic heterogeneity of human breast cancer has long been recognized. Now, molecular profiling has enriched our understanding of breast cancer heterogeneity and yielded new prognostic and predictive information. Despite recent therapeutic advances, including the HER2-specific agent, trastuzumab, locoregional and systemic disease recurrence remain an ever-present threat to the health and well being of breast cancer survivors. By definition, disease recurrence originates from residual treatment-resistant cells, which regenerate at least the initial breast cancer phenotype. The discovery of the normal breast stem cell has re-ignited interest in the identity and properties of breast cancer stem-like cells and the relationship of these cells to the repopulating ability of treatment-resistant cells. The cancer stem cell model of breast cancer development contrasts with the clonal evolution model, whereas the mixed model draws on features of both. Although the origin and identity of breast cancer stem-like cells is contentious, treatment-resistant cells survive and propagate only because aberrant and potentially druggable signaling pathways are recruited. As a means to increase the rates of breast cancer cure, several approaches to specific targeting of the treatment-resistant cell population exist and include methods for addressing the problem of radioresistance in particular.

Targeted Therapies for Triple-Negative Breast Cancer: Combating a Stubborn Disease

Triple-negative breast cancers (TNBCs) constitute a heterogeneous subtype of breast cancers that have a poor clinical outcome. Although no approved targeted therapy is available for TNBCs, molecular-profiling efforts have revealed promising molecular targets, with several candidate compounds having now entered clinical trials for TNBC patients. However, initial results remain modest, thereby highlighting challenges potentially involving intra- and intertumoral heterogeneity and acquisition of therapy resistance. We present a comprehensive review on emerging targeted therapies for treating TNBCs, including the promising approach of immunotherapy and the prognostic value of tumor-infiltrating lymphocytes. We discuss the impact of pathway rewiring in the acquisition of drug resistance, and the prospect of employing combination therapy strategies to overcome challenges towards identifying clinically-viable targeted treatment options for TNBC.

Determination of Somatic and Cancer Stem Cell Self-Renewing Symmetric Division Rate Using Sphere Assays

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cells resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence.

Spontaneous apoptosis of blood dendritic cells in patients with breast cancer.

IntroductionDendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.MethodsSpontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.ResultsIn this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglandin (PG)E2 as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.ConclusionCumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.

Response of Estrogen Receptor-Positive Breast Cancer Tumorspheres to Antiestrogen Treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

Low Level Plasmodium falciparum Blood-Stage Infection Causes Dendritic Cell Apoptosis and Dysfunction in Healthy Volunteers

BACKGROUND Dendritic cells (DCs) are highly specialized antigen-presenting cells that are crucial for initiation of immune responses. During naturally acquired malaria, DC number and function is reduced. METHODS The timing of, parasitemia threshold of, and contribution of apoptosis to DC loss were prospectively evaluated in 10 men after experimental challenge with approximately 1800 Plasmodium falciparum-parasitized red blood cells (pRBCs) and after drug cure initiated at a parasite level of ≥ 1000 parasites/mL. RESULTS The nadir levels of total, myeloid, and plasmacytoid DCs occurred 8 days after infection. DC loss was partially attributable to apoptosis, which was first detected on day 5 (median parasite level, 238 parasites/mL) and maximal at day 7. Remaining DCs exhibited a reduced ability to uptake particulate antigen. DC numbers recovered approximately 60 hours after antimalarial drug administration. There was no loss of DC number or function before or after drug cure in 5 men inoculated with <180 pRBCs and treated on day 6, when their parasite level was approximately 200 parasites/mL. CONCLUSIONS Plasmodium causes DC loss in vivo, which is at least partially explained by apoptosis in response to blood-stage parasites. In primary infection, loss of DC number and function occurs early during the prepatent period and before or with onset of clinical symptoms. These findings may explain in part the inadequate development of immunity to blood-stage malaria infection.