Mechanisms of immune evasion in breast cancer

Abstract Tumors develop multiple mechanisms of immune evasion as they progress, with some cancer types being inherently better at ‘hiding’ than others. With an increased understanding of tumor immune surveillance, immunotherapy has emerged as a promising treatment strategy for breast cancer, despite historically being thought of as an immunologically silent neoplasm. Some types of cancer, such as melanoma, bladder, and renal cell carcinoma, have demonstrated a durable response to immunotherapeutic intervention, however, breast neoplasms have not shown the same efficacy. The causes of breast cancer’s immune silence derive from mechanisms that diminish immune recognition and others that promote strong immunosuppression. It is the mechanisms of immune evasion in breast cancers that are poorly defined. Thus, further characterization is critical for the development of better therapies. This brief review will seek to provide insight into the possible causes of weak immunogenicity and immune suppression mediated by breast cancers and highlight current immunotherapies being used to restore immune responses to breast cancer

Hepatocyte Growth Factor, a Determinant of Airspace Homeostasis in the Murine Lung

<div><p>The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor <em>Met</em> in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF–mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.</p> </div

HGF treatment improves airspace caliber in TSK/+ mice.

<p>A. Serum HGF levels in mice treated with HGF infusion pumps at 50 µg/d. **p<.001 B. Phospho-Met immunofluorescent staining in lungs of mice treated with HGF infusion compared with PBS carrier infusion. Arrow in inset denotes phosphorylated c-Met (p-Met, green) in alveolar epithelial cells of HGF treated mice. Scale bar: 50 µm. C. Histology of TSK/+ lung treated with HGF compared with untreated controls. 20× magnification. Scale bar 100 µm. D. Morphometric assessment of airspace dimension in mice treated with HGF for 2 weeks compared with wild-type mice and untreated controls. Lo HGF-50 µg/d. Hi HGF-100 µg/d. E. Representative staining for oxidative stress marker nitrotyrosine in lungs of TSK/+ mice treated with HGF and controls. Arrow denotes staining in alveolar epithelial cells. 40× magnification. Scale bar 50 µm. F. Quantitative immunohistochemistry of nitrotyrosine staining in TSK/+ lungs treated with saline or HGF by infusion pump compared to wild-type controls. G. Representative immunoblotting of normalized phosphomediator levels (akt and stat3) in two TSK/+ mice treated with HGF compared with saline treated mice. N = 4–8 mice per group and treatment.</p

Generation and characterization of mice deficient in <i>Met</i> expression in alveolar epithelial cells.

<p>A. Right panel-Representative immunohistochemical staining of c-Met in 2 week old mouse lungs. 40× magnification. N = 4–6 mice. Arrows denote expression in type II epithelial cells. Left panel-Representative immunohistochemical staining for HGF in 2 week old mouse lungs. 40× magnification. N = 4–6 mice. Scale bar (L): 25 µm, (R): 50 µm. Arrows denote exclusion of HGF from alveolar epithelial cells with apparent localization to the interstitium. B. Representative fluorescent immunohistochemistry of phosphorylated c-Met in mice deficient in <i>Met</i> and bitransgenic controls. Green-p-Met. 40× magnification. N = 4–6 mice. Scale bar: 25 µm. C. Quantitative immunohistochemistry of p-met expression in the airspace of <i>Met</i>-deleted mice and controls. D. Representative histology of mice deficient in airspace <i>Met</i> expression and controls at two weeks of age. Note patchy airspace enlargement in the targeted mice. Scale bar: 100 µm. E. Airspace dimension by morphometry in <i>Met</i>-deficient mice and controls at 2 and 3 weeks of age. F. Quantitation of SPC+ cells in the airspace by SPC immunohistochemistry in <i>Met</i>-deficient mice compared with control bitransgenic mice. *p<0.05. G. Representative thrombomodulin immunohistochemical staining of the microvascular bed in the lung parenchyma of Met deficient mice compared with controls. Inset shows reduced staining in the alveolar epithelial walls. 40× magnification, inset 100×. N = 5–7 mice per genotype. H. Quantitative immunohistochemistry of thromobomodulin staining of <i>Met</i>-deficient mice and controls. **p<0.01.</p

HGF treatment of MLE12 induces prosurvival signaling that protects against alveolar cell death.

<p>A. Representative immunoblots of phosphoproteins in mice treated with HGF for 5 min (pERK and pJNK) or 15 min (pAKT) showing dose response. B. Cleaved caspase 3 immunoblotting in MLE12 cells treated with staurosporine with or without HGF or wortmannin. All experiments performed in triplicate.</p

Increased oxidative stress, reduced alveolar cell proliferation, and increased inflammation in lungs of <i>Met</i>–deficient mice.

<p>A. Representative immunohistochemical staining for proliferation marker Ki67 in <i>Met</i>-deficient mice compared with controls. Arrows show increased alveolar cell proliferation in control lungs compared with mutant lungs. Arrowheads denote increased proliferation signal in alveolar macrophages in mutant lungs. Scale bar: 50 µm (top panel), 25 µm (bottom panel). B. Quantitative immunohistochemistry of Ki67 staining in the alveolar epithelium of <i>Met</i>-deficient mice and controls. C. Representative immunohistochemical staining (brown) for nitrotyrosine (NiTyr) in lungs of control and <i>SPCMet</i>^f/f mice. Arrowheads denote positive staining. 20× magnification. N = 4 mice per genotype. Scale bar: 50 µm. D. Quantitative immunohistochemistry of nitrotyrosine staining in <i>SPCMet^f/f</i> mice compared with controls. E. Macrophage abundance per Mac3 immunohistochemistry in <i>Met</i>-deficient lungs and controls at 2 and 3 weeks of age. Note increasing macrophage influx in mutant lungs. N = 4–6 mice per genotype. F. Representative immunohistochemical staining (brown) for macrophages (Mac3) in lungs of control and <i>SPCMet ^f/f</i> mice at 3 weeks of age. Arrowheads denote positive staining. 40× magnification. N = 4–6 mice per genotype. Scale bar: 25 µm.</p

HGF signaling induces proliferation and scattering of MLE12 cells.

<p>A. Proliferation dose response of HGF and EGF treatment of MLE12 cells demonstrating a significant induction of proliferation by HGF. *p<0.05. B. Proliferation response of MLE12 cells transfected with <i>MET</i> or a control vector showing increased proliferation resulting from <i>MET</i> transfection. C. Cell dispersion images of MLE12 cells treated with EGF or HGF compared to media control. D. Effect of HGF treatment on staurosporine induced caspase 3 cleavage. ST-staurosporine. All cell experiments performed in triplicate. CC3-cleaved caspase 3. E. Densitometric quantitation of effect of HGF treatment on staurosporine induced caspase 3 cleavage. F. Survival time-course of primary alveolar epithelial cells from control and <i>Met</i>-deficient mice. *p<0.05. G. Relative expression of <i>Gclc</i> and <i>Nqo1</i> with HGF treatment of primary murine alveolar epithelial cells. *p<0.05 compared with control conditions.</p