Peroral treatment with E and E + P4 after OVX results in similar tumor incidence but more adenocarcinomas following NMU relative to Sham.

<p>(a) Rats were OVX on PND 40, injected with NMU on PND 50, and palpated biweekly for the appearance of tumors. There was no difference in tumor incidence between E, E + P4 and Sham but all three groups were different from P only and OVX, which were not different from one another (p < 0.05, Mantel-Cox Log-rank test). (b) Tumor type was classified by a histological pathologist as adenoma or adenocarcinoma based on the most malignant part of each section. The effect of treatment on tumor type was analyzed using a Chi-square analysis (* p < 0.05). (c) Tumors were measured with a Vernier caliper the morning of sacrifice. To calculate tumor burden, the volume of each tumor was added together per animal. There were no differences in tumor burden among treatment groups.</p

Serum E2 levels are elevated with peroral hormone treatment.

<p>Blood samples were drawn from the lateral tail vein 5, 3, and 5 hours after treatments were presented 4, 8, and 12 weeks after NMU injection, respectively. Blood draws from all animals were completed within 2 hours on each of the three days. Serum was analyzed using an E2 ELISA (Calbiotech). Blood from one animal in the 12-week E only group and one animal in the 8-week E + P4 group hemolyzed, thus these samples were not included in the analysis. All values from E and E + P4 animals are presented to demonstrate the range of serum E2 across animals and treatments while a subset of Sham animals and serum pools from OVX animals were included for comparison. Horizontal lines indicate mean values for each time point.</p

Long-term daily peroral E treatment is physiologically active.

<p>At sacrifice, uteri were excised and weighed. Images of uteri were taken before storage in 10% NBF. (a) Representative images of uteri from each treatment group. (b) Uterine weights were reduced in OVX rats treated with vehicle but increased with E or E + P4 treatment. P4 alone was unable to restore uterine weights. Uterine weights are expressed as mean ± SEM; n = 8, 7 for P4 only; p < 0.05, one-way ANOVA; Newman-Keul’s posttest; different letters denote significant difference. (c) Rats were weighed biweekly for the duration of the study. Long-term exposure to E or E+ P4 resulted in body weights that were similar to those of Sham controls. Daily treatment with P4 did not prevent weight gain following OVX. Body weights are expressed as mean ± SEM, n = 8, 7 for P4 only; repeated measures ANOVA through the first animal sacrifice at 13 weeks post NMU (PND 148) (treatment: p < 0.01) with Bonferroni-Dunn multiple comparisons test (* start of p < 0.05 for OVX, # start of p < 0.05 for P4 only; compared to Sham). The pattern persisted in the remaining animals at the end of the study (p < 0.05, one-way ANOVA, Newman-Keul’s posttest).</p

Treatment of OVX animals with E or E + P4 results in tumors with similar proliferation indices and receptor status relative to tumors from Sham controls.

<p>Ki67, ERα, and PR IHC was performed and measured as described. Images depicting low (a, d, g) and high (b, e, h) expression of Ki67, ERα, and PR are presented with corresponding scores in the bottom left corner of each image. There were no differences in Ki67 or ERα expression between treatment groups. Animals treated with E + P4 had more PR expression than animals treated with E alone (Mann-Whitney test comparing E only to E + P4, p < 0.05).</p

Treatment with E leads to increased uterine metaplasia in treated animals.

<p>Cross sections of uteri were stained with hematoxylin and eosin. A toxicological pathologist staged and graded the slides from 0–3 for each category leading to scores of stage + grade, specifically focusing on the uterine lumen (arrows) and not on dilated ducts (arrow heads). Images are representative of 0+0 (relatively normal), 1+1, 2+2, and 3+3 respectively. The larger images were taken at 20X magnification and the inset at 4X magnification. Scores were derived by adding stage and grade to get a final score of 0–6. P4 only and OVX animals were not included in analysis as all uteri were atrophied and not metaplastic. Uterine scores are expressed as mean ± SEM; n = 8; p < 0.05, Kruskal-Wallis test with Dunn’s Multiple Comparison’s posttest; different letters denote significant difference.</p

Mammary gland development of E and E + P4 treated animals is similar to Sham controls.

<p>Representative images of whole mounts (4X magnification) demonstrated ductal thickness was similar in Sham (a), E only (b), and E + P4 (c) while the overall morphology of glands was similar between OVX (d) and P4 only (e) with thinner ducts relative to the other groups. The length of the mammary gland parenchyma was measured from the lymph node to the most distal edge of the ducts using FIJI as shown in (f). The parenchyma from Sham, E only, and E + P4 glands exhibited similar ductal lengths while OVX and P4 only were shorter (g). Mammary gland length is expressed as mean ± SEM; p < 0.05, one-way ANOVA; Newman-Keul’s posttest.</p

Investigating ICI and Lapatinib induced matriptase determined that only modest activation resulted from PI3K inhibition with no activation from MAPK inhibition and occurs as early as six hours post treatment.

A) BT474 and ZR-75-30 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then treated with 5.4uM Lapatinib, 10uM Trametinib, 10uM Pictilisib, or 0.01% DMSO. Conditioned media was collected with the appropriate lysis buffer (see methods). B) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then spiked with 5.4uM Lapatinib for increasing amounts of time. Cell lysates and conditioned media were collected with the appropriate lysis buffer. The levels of phospho-AKT, total and activated matriptase were determined by immunoblotting with p-S473 AKT and total matriptase (M24) monoclonal antibodies. C) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for increasing amounts of time in serum-free media with 1uM ICI. After each time point, cells were spiked with 5.4uM Lapatinib for 1 hour. Cell lysates were collected with the appropriate lysis buffer and the levels of total and activated matriptase were determined by immunoblotting with the total matriptase (M24) monoclonal antibody. GAPDH was used as a loading control. Panels were the highest quality representative of 4 biological replicates.</p

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Investigating matriptase-directed 5mM NAC treatment resulted in increased apoptosis alone or in combination with ICI and/or Lapatinib in ER-positive breast cancer models.

100,000 A) BT474, B) MDA-MB-361 and C) MCF7 cells /well were seeded in 6-well plates and allowed to attach overnight. Cells were then treated with 5mM NAC, 5.4 uM Lapatinib, and 1uM ICI alone or in combination in serum-replete media for four days. NAC was administered again on day 3. Cells were collected and apoptosis determined by Annexin V/Propidium Iodide staining. Graphs are representative of 3 biological replicates. BT474 control (**, p = .0017 vs NAC), Lap (**, p = .0013 vs Lap+NAC), Lap+ICI (**, p = .0044 vs triple), MDA-MB-361 ICI (**, p = .0014 vs ICI+NAC), Lap (**, p = .0028 vs Lap+NAC), Lap+ICI (*, p = .0311 vs triple) MCF7 control (**, p = .0055 vs NAC), ICI (**, p = .0096 vs ICI/NAC), Lap/ICI (***, p = .0001 vs triple). Data shown as mean +/- SD of triplicate experiments;* p<0.05, ** p<0.01, ***p<.0.001.</p

Investigating endogenous, cell stress, and acid-induced matriptase activation demonstrated those mechanisms were mediated ROS and could be inhibited by N-acetyl cysteine (NAC).

A) SKBR3 cells were seeded in 35mm dishes and when ~80% confluent, washed twice with 1X sterile PBS and incubated for 3 hours with 5.4uM Lapatinib. Cells were then spiked with 10nm hydrogen peroxide for increasing amounts of time. Conditioned media was collected from the cells and analysed for levels of total and activated matriptase by the total matriptase (M24) antibody. B) MCF7 and C) BT474 cells were seeded in 35mm dishes and when 80% confluent. 5mM NAC was added to serum-replete media for 3 hours to pre-treat the cells. After pre-treatment, acid-induction buffer and 5mM NAC were added to cells either alone and in combination for 20 minutes. Cell lysates and conditioned media were collected in parallel and assayed for GAPDH as well as total and activated matriptase by the GAPDH and total matriptase (M24) antibody. D) MDA-MD-468, AU565, and SKBR3 cells were seeded in 35mm dishes and when ~80% confluent, incubated in 1ml serum-replete media with and without 5mM NAC for 24 hours. Conditioned media was collected from the cells and analysed for levels of total and activated matriptase by the total matriptase (M24) and activated matriptase (M69) antibody. Panels were representative of 3 biological replicates. E) AU565 cells were seeded in 35mm dishes and ~80% confluent, incubated in 1ml serum-replete media. Conditioned media was collected and 5mM NAC was added to experimental samples for 24 hours. The levels of activated matriptase were then assessed using total and activated matriptase antibodies. Panels were the highest quality representative of 4 biological replicates.</p