α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFß antibody to promote durable rejection and immunity in squamous cell carcinomas.

BACKGROUND: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFß signaling. METHODS: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFß or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. RESULTS: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFß monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFß, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFß antibody administration attenuates these effects. We show that α-TGFß acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFß acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. CONCLUSIONS: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFß-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFß combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFß/α-PD-1 combination therapy (NCT02947165).

Tyrosine phosphatase PTPα contributes to HER2-evoked breast tumor initiation and maintenance.

Protein tyrosine phosphatase alpha (PTPα/PTPRA) was shown previously to be overexpressed in human primary breast cancers, and to suppress apoptosis in estrogen receptor-negative breast cancer cells in vitro. However, it is not known whether PTPα is important for mammary tumor initiation, maintenance and/or progression. We have used a combination of three-dimensional cultures, a transgenic mouse model of breast cancer lacking PTPα as well as xenografts of human breast cancer cell lines to address these questions. We found that PTPα knockdown after overt tumor development reduced the growth of HER2-positive human breast cancer cell lines, and that this effect was accompanied by a reduction in AKT phosphorylation. However, PTPα knockdown did not affect invasiveness of HER2-positive human breast cancer cells grown in three-dimensional cultures. Moreover, in MMTV-NeuNT/PTPα(-/-) mice, PTPα ablation did not affect NeuNT-evoked tumor onset or metastasis but decreased the number of tumors per mouse. Thus, we demonstrate that PTPα contributes to both HER2/Neu-mediated mammary tumor initiation and maintenance. Our results suggest that inhibition of PTPα can have a beneficial effect on HER2-positive breast cancers, but that inhibition of additional targets is needed to block breast tumorigenesis.

Expression of PIK3CA mutant E545K in the mammary gland induces heterogeneous tumors but is less potent than mutant H1047R.

The phosphoinositide 3-kinase (PI3K) signaling cascade is a key mediator of cellular growth, survival and metabolism and is frequently subverted in human cancer. The gene encoding for the alpha catalytic subunit of PI3K (PIK3CA) is mutated and/or amplified in ∼30% of breast cancers. Mutations in either the kinase domain (H1047R) or the helical domain (E545K) are most common and result in a constitutively active enzyme with oncogenic capacity. PIK3CA(H1047R) was previously demonstrated to induce tumors in transgenic mouse models; however, it was not known whether overexpression of PIK3CA(E545K) is sufficient to induce mammary tumors and whether tumor initiation by these two types of mutants differs. Here, we demonstrate that expression of PIK3CA(E545K) in the mouse mammary gland induces heterogenous mammary carcinomas but with a longer latency than PIK3CA(H1047R)-expressing mice. Our results suggest that the helical domain mutant PIK3CA(E545K) is a less potent inducer of mammary tumors due to less efficient activation of downstream Akt signaling.

Patient care conference: a coordinated, collaborative effort.

The flow chart, guidelines, and document describe the mechanism for patient care conferences. Because the need, process, and documentation of patient care conferences have been clarified, care conferences are now more consistent, efficient, and effective. Documentation streamlined the process of setting up care conferences by recording the following information: who was notified, who would attend, specific issues to be discussed, conference scheduling summary of plans and decisions made. Task force satisfaction was high because of a short timeline, clear problem definition, clear goals, team sponsorship, and a good end product. Use of continuous quality improvement techniques added to the success of this project, because there was a methodology for starting with a variable, unclear process and ending with a creative, efficient process. All members of the healthcare team and patients and families were satisfied, because the patient care conference was available to discuss specific issues and reach consensus on decisions about the patient's treatment plan at critical points during the patient's hospital course.

The granular epithelioid cells in the kidney of the lemon sole (Pleuronectes microcephalus Donovani).

Serial sections from kidneys of 5 aglomerular lemon soles (Pleuronectes microcephalus) demonstrated the presence of anastomosing arteriolar networks in the caudal half of the organs. There was no preferred location of the networks, which were found both near the surface and in the deeper parts of the kidney. The size of the networks varied; the largest measured more than 900 micrometers in the longest axis and covered an area larger than 500,000 micrometers2, whereas the smallest measured about 60 micrometers in diameter with an area of 2040 micrometers2. The larger networks were invariably located close to large intrarenal veins. Anastomoses were found between the arteriolar networks and the peritubular capillaries and also associated with veins. The walls of the arterioles were composed of granulated epithelioid cells, exhibiting numerous intracytoplasmic granules. These granules occasionally had a rod-like appearance with a paracrystalline substructure. Using antibodies directed against murine and human renin, the epithelioid cells in the walls of the arteriolar networks and the cytoplasmic granules revealed a positive immunoreaction. The results suggest that the renin-angiotensin system in the aglomerular lemon sole may be important for both the regulation of the systemic blood pressure and the blood supply to the peritubular capillaries of the kidney.

Juxtaglomerular apparatus of the human kidney: correlation between structure and function.

Electron microscopic and morphometric evaluation of the juxtaglomerular apparatus (JGA) of the human kidney showed the following: (1) The JGA of the human kidney consists of the epithelioid cells of afferent and efferent arterioles, the Goormaghtigh cells, and the macula densa, and it is abundantly supplied with sympathetic nerves. (2) The macula densa is in contact with the juxtaglomerular cell complex (JGC), the endocrine part of the JGA, via the Goormaghtigh cell field. The surface area of the macula densa is much larger than is the contact area with the JGA, totalling, in the healthy human kidney, some 66 cm2. (3) The total volume of all JGC's is 26 to 40 mm3. (4) Morphometric investigations of the JGC showed that appropriate stimulation by a decrease in the intrarenal or systemic blood pressure led to hypertrophy and hyperplasia of the JGC, which, in extreme cases, may also lead to a transformation of both the Goormaghtigh cells and mesangium cells into epithelioid cells.

The normal juxtaglomerular apparatus in the human kidney. A morphological study.

Out of 49 serially studied juxtaglomerular apparatuses, 6 typical variants from two normal human kidneys were reconstructed graphically. The agranular Goormaghtigh cells filled the entire space between the macula densa, the afferent and the efferent arterioles and the glomerular mesangium. The Goormaghtigh cells were always in direct contact with all the other structures. They also invariably continued into the glomerular mesangium. The distal tubule regularly showed widening in the macula densa segment and, at this level, there was considerable variation in the shape of the distal tubule. Direct contact between the macula densa and the hilar arterioles was not always present, the area of contact was usually greater with the afferent than with the efferent arteriole.

The juxtaglomerular apparatus in a human kidney with polar artery stenosis.

Juxtaglomerular apparatuses (JGA) of a human kidney with stenosis of a polar artery from a hypertensive 18-year-old male patient were studied qualitatively and quantitatively on 2 mu thick serial sections from plastic embedded renal tissue. 11 JGA from juxtamedullary and 9 JGA from subcapsular cortical zones were photographed serially, the copies taped together and the cells and relationships within the JGA studied. On the copies the lenght of contact between the different juxtaglomerular structures and the basement membrane of the macula densa was measured. Futhermore we calculated the areas of surface contact and the macula densa basal area. In the juxtamedullary JGA affected by the polar artery stenosis all Goormaghtigh cells were transformed into epitheloid cells and the Goormaghtigh cell field was significantly larger than in the JGA of the subcapsular cortex, which obviously had been exposed to the systemic hypertension. The macula densa basal area was significantly greater in the juxtamedullary JGA than in the subcapsular JGA, but neither of these differed significantly from the macula densa in the normal JGA reported previously.

The size of the juxtaglomerular apparatus in diabetic glomerulosclerosis and its correlation with arteriolosclerosis and arterial hypertension: a morphometric light microscopic study on human renal biopsies.

The surface areas of the juxtaglomerular cell complexes and the index of arteriolar sclerosis were evaluated in renal biopsies from 50 patients with diabetic glomerulosclerosis. The mean surface areas of the JGC's were significantly enlarged compared to that of normal renal tissue, whereas no significant difference could be found between kidneys from hypertensive and the normotensive diabetics. A positive linear correlation was found between mean JGC-size and the index of sclerosis of the afferent arterioles. This was interpreted as the result of faulty coordination between the activity of the juxtaglomerular apparatus and the systemic arterial blood pressure. This is caused by stenoses of the preglomerular afferent arterioles in diabetic arteriosclerosis.

The structure of the juxtaglomerular apparatus in Addison's disease, Bartter's syndrome, and in Conn's syndrome: a comparative, morphometric, light microscopic study on serial secions.

Continuing and supplementing previous morphometric studies on the juxtaglomerular apparatus (JGA) of normal kidneys we have now investigated semi-thin serial sections of each 10 hyperplastic and hypertrophied JGAs in Addison's disease and in Bartter's syndrome, as well as 8 atrophic JGAs in Conn's syndrome. With the exception of Bartter's syndrome, where in only two out of ten JGAs the efferent arteriole, and in none of them the afferent arteriole touches immediately the macula densa, there is an almost regular direct contact between the hilar arterioles and the macula densa like in normal kidneys. The Goormaghtigh cell field invariably touches the macula densa. In Bartter's syndrome, but not in Addison's disease, a considerable enlargement of the macula densa was measured, associated with an exceptional enlargement of the Goormaghtigh cell field. In all cases examined here about 40-60% of the basal area of the macula densa do not have any direct contact with other structures forming the JGA.

The structure of the human juxtaglomerular apparatus. A morphometric, lightmicroscopic study on serial sections.

49 juxtaglomerular apparatuses were examined using 1.9mu thick Giemsa-stained serial sections from human renal tissue embedded in plexiglass. In 43 juxtaglomerular apparatus the direct contact areas between the different juxtaglomerular structures and the basal area of the macula densa were calculated. A positive, significant correlation was found between the size of the macula densa and the direct contact area between macula densa and Goormaghtigh's cell field on the one hand, and between the macula densa and the direct contact area between Goormaghtigh's cell field and the afferent arteriole on the other. There was also a significant, positive correlation between the direct contact area of Goormaghtigh's cell field with the macula densa and that of Goormaghtigh's cell field with the afferent arteriole. On the efferent side none of these correlations were significant. Thus a "flow of information" from the macula densa via the Goormaghtigh cells to the afferent arteriole is morphologically possible. The direct contact areas between macula densa and the afferent or the efferent arterioles were not correlated with any of the other parameters. Epithelioid cells were present in the interlobular arteries, prior to and within the juxtaglomerular apparatus in the afferent arterioles, as well as within and beyond the juxtaglomerular apparatus in the efferent arterioles.