Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting

Abstract

Background: Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models.

Methods: Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin.

Results: TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice.

Conclusions: TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.

Fig 1. Expression of TfR in primary breast tumors.

A-C) Gene expression of TFRC A) by stage (TGCA, total N = 1192), B) by histological subtype (GENT2, total N = 1467), C) by grade (GENT2, total N = 725). D-H) TfR immunohistochemistry (IHC) D) H-score of normal breast (outside of tumor margin, N = 8) versus malignant breast tumors (N = 99), E) representative images of breast cancer histological subtypes, F) H-score by subtype (total N = 98), G) H-score by stage (N = 99), H) H-score by grade (N = 91). *P<0.05 T-test, ***P<0.001, ****P<0.0001 one-way ANOVA-Tukey’s post-hoc test.

Fig 2. Expression of TfR after neoadjuvant therapy.

A) experimental design: unless otherwise specified pre- refers to tissue from the diagnostic biopsy, post- refers to tissue from the resected tumor; B) TFRC expression in GSE21974: 32 patients sampled before and after four cycles of epirubicin and cyclophosphamide prior to taxane (EC-T), C) TFRC expression in GSE18728 (N = 22 patients): biopsy samples collected at diagnosis (Pre) and after one cycle of capecitabine and docetaxel (NAC: Cy-TX), and from the resected tumor (Res); D) TFRC expression in GSE114082 (N = 17 patients) pre- and after trastuzumab (NAC:TR). E-G) Tumor microarray IHC TfR score E) waterfall plot of patient-matched ER+ breast cancer (BC) before and after NAC (N-14), F-G) H-score in (non-matched) samples from diagnostic punch biopsy (Pre, grey), resected tumors (Post-NAC, red), and regional lymph nodes (R-LN, teal) from F) ER+ patients (N = 38) and G) TNBC patients (N = 35. H) TfR IHC of PDX WHIM68, treated with paclitaxel (30mg/kg) and carboplatin (50mg/kg) (Carbotaxol) versus vehicle, and harvested after treatment and at relapse; treatment scheme, representative images of TfR staining and quantification scatter plot post-vehicle, post-NAC, and at relapse. *P<0.05 by paired T-test, **P<0.01 by ANOVA and Tukey’s post-hoc test.

Fig 3. TfR expression in breast cancer metastases.

A) TFRC expression in GSE175692 biopsies from different metastatic sites (total N = 117), B) TFRC expression in GSE43837 of matched primary breast cancer and brain metastases samples (N = 18 patients). C-G) TMA TfR staining of primary breast cancer and bone metastases C) representative image, D) waterfall plot of 28 patient-matched primary tumor and bone metastasis samples (black breast, red bone); E-G) previous treatments at the time of metastasis in TMA samples: No Tx = no treatment, C = chemotherapy, C+E = chemotherapy and endocrine therapy, R = radiation therapy, R+C = radiation and chemotherapy, R+E = radiation and endocrine therapy, R+C+E = radio- chemio- and endocrine therapy, E) tumor subtypes vs treatments, LumA = luminal A, LumB = luminal B, HER2 = Her2-high, TNBC = triple negative breast cancer, cells: number, F) H-Score of bone metastases by treatment (tot N = 24). G) H-Score of bone metastases by histological subtype (tot N = 25).

Fig 4. Biodistribution of AF680-Tf in 4T1 metastatic model versus non-tumor Balb/c mice.

A) In vivo imaging over time relative to injection of AF680 in one non-tumor (top) and two tumor bearing mice (bottom), left: BLI at t = 24h tumor sites, right: PerL imaging, B) ex-vivo PerL imaging for AF680 of lungs, kidneys (K), liver, and spleen (Sp) of normal (left) versus 4T1 tumor-bearing mice (right); C-D) AF680-Tf fluorescence intensity (F.I.) in arbitrary units (A.U.) of group 1 organs of non tumor (grey, NoT) versus tumor (red, 4T1) mice at C) 6h from injection (N = 4 non tumor and N = 5 tumor) and D) 24h from injection (N = 3 non tumor, N = 4 tumor); E) representative ex-vivo BLI (top) and AT680-Tf imaging (bottom) of hindlimb bones (Leg) and spine of one non-tumor (left) and two 4T1 tumor bearing mice (right); F-G) AF680-Tf fluorescence intensity (F.I.) in arbitrary units (A.U.) of bones of non tumor (grey, NoT) versus tumor (red, 4T1) mice at F) 6h from injection (N = 4 non tumor and N = 5 tumor) and G) 24h from injection (N = 3 non tumor, N = 4 tumor). **P<0.01 by two-way ANOVA and Šidák test.

Fig 5. Non-toxic iron chelation upregulates transferrin uptake in breast cancer cells in vitro but does not affect tumor growth in vivo.

A-D) In vitro uptake of transferrin conjugates in breast cancer cells A) live imaging of Phrodo Red transferrin conjugate uptake MDA-MB-231 treated with vehicle (left) or DFO 200μM for 48h (right); B) uptake of Tf by fluorimetry of MDA-MB-231 cells pre-treated with FAC 200μM, or DFO 200μM for 48h, ex485/em515nm fluorescence divided by 570 nm optical density after nuclear staining with crystal violet (CV), C) T47D cells pre-treated with FAC 200μM, or DFO 200μM for 24h, ratio of fluorescence intensity at ex485/em515 nm (AF488-Tf) and Hoechst (ex350/em461 nm). D) AF488-Tf uptake by Bo1 cells treated with 100 μM DFO or FAC vs vehicle (AF488-Tf/CV as above); No Tf: background control of cells not exposed to fluorescent transferrin; *P<0.05, ***P<0.001, ****P<0.0001, ns non significant; one-way ANOVA and Tukey’s post-hoc test. E-F) Effect of treatment with DFO in vivo on serum ferritin in mice, E) treatment diagram: non-tumor bearing mice (N = 5) were injected i.m. six times over 11 days with vehicle (HBSS), DFO 100mg/kg, or DFO 200mg/kg, and i.v. AF750-Tf on day 11, then sacrificed at day 12 F) serum ferritin ELISA at day 12, black dashed line: normal range, red dashed line: abnormal or pharmacologically lowered ferritin. G, H) Effect of DFO treatment on tumor growth in the metastatic Bo1 model G) treatment diagram: mice were inoculated by left ventricle injection, then imaged by BLI to establish tumor engraftment at day 4 and growth at day 8 and 11, treated with 4 injections of DFO 200mg/kg over 5 days, and intravenous AF750-Tf on day 10, H) total-body BLI at 4, 8, and 11 days after intracardiac inoculation of Bo1, average radiance in p/s/cm²/sr (N = 5/group).

Fig 6. Iron chelation in metastatic Bo1 enhances the uptake of transferrin (AF750) at tumor sites.

A-B) In vivo imaging with BLI and AF750 A) representative images of two tumor-bearing mice treated with vehicle (left) and two with 4 i.p. injections of DFO 200mg/kg (right), by BLI (top, rainbow pseudocolor of average radiance) and 750 nm epifluorescence (bottom, blue-hot psuedocolor of average radiant efficiency), red arrows: tumor lesions seen by BLI but not AF750, geen arrows: tumor sites where BLI and AF750-Tf co-localize, B) percentage of tumor foci by BLI that did (yellow) or did not (purple) show AF750-Tf accumulation in mice treated with vehicle versus DFO, P<0.0001 by Fisher’s exact test. C-J) ex-vivo imaging by BLI and AF750, representative images of BLI (top) and 750nm epifluorescence (bottom) (C, G, H), correlation between BLI and AF750 (E,F, I, J), bar graphs (G,K), C) example of group 1 organs ex-vivo imaging in vehicle (top) and DFO—treated (bottom) mice; D) representative image of adrenal glands, mesenter (fat, vessels, and lymph nodes), ovarian omentum, ovaries, uterus and tubae in vehicle (left) and DFO -treated (right) mice; E-F) correlation between tumor burden and AF750-accumulation in ROI from group 2: X axis, Log-transformed average radiance (photons/s/cm²/sr), Y axis, Log-transformed average radiant efficiency ((photons/s/cm²/sr)/(μW/cm²) E) vehicle-treated mice and F) DFO-treated mice; G) comparison of AF750-Tf uptake in the legs and spines of non tumor-bearing (N = 3, grey bars, No Tumor), Bo1 mice injected with vehicle (N = 5, teal), and Bo1 mice treated with DFO (N = 5, red); **P<0.01, ****P<0.0001 by two-way ANOVA and Šidák test; H) representative images of spines from two vehicle-treated (left) and two DFO-treated (right) BO1 mice I-J) correlation between tumor burden and accumulation of Tf in the bones (pelvis, legs, spine) of I) vehicle-treated (P = 0.0074, Pearson’s r = 0.522, R = 0.272) and J) DFO-treated (P<0.0001, r = 0.769, R = 0.591); K) percentage of tumor foci by ex-vivo BLI that did (yellow) or did not (purple) show AF750-Tf accumulation in mice treated with vehicle versus DFO, P = 0.006 by Fisher’s exact test.

Fig 7. Iron chelation enhances the uptake of transferrin (AF750) in low-tumor burden Bo1 model.

A) Experimental design: mice were inoculated with Bo1 i.c. and treated from day 3 imaged with vehicle (V) or DFO; injection of AF750-Tf on day6 and imaging day 7; B) quantification of total-body radiance over time show no difference in tumor bearing mice treated with vehicle or DFO, *** P<0.001 in non tumor-bearing controls C) ex-vivo imaging of bones from one vehicle and one DFO-treated mice by BLI (top) and AF750-tf epifluorescence (bottom), D-E) Correlation between BLI and AF750-Tf fluorescence in the bones (legs, pelvis, spine) of D) vehicle-treated mice (P = 0.069, r = 0.42, R = 0.17, N = 5) and E) DFO-treated (P<0.0001, r = 0.66, R = 0.43, N = 5) mice.