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Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 3 (HER3) has rapidly gained much attention as a promising target for cancer treatment. The increasing recognition of HER3 roles in a number of HER family-driven cancers has led to studies aimed at targeting this receptor and developing HER3-targeted platforms with the ability to deliver therapeutic genes. We have previously indicated that the flexible linker and one unit of RALA in affibody-based platform could target HER3 and deliver its cargo. Based on the previous finding, in a new class of affibody-based platforms, we used two different linkers and RALA units and then compared their effectiveness on targeting and delivering specified genes to HER3 positive cells. Our results clearly showed that our biopolymeric platforms can successfully condense DNA into nanoparticles and object the overexpressed HER3 receptors and then transfer specific genes. Our affibody-based platform containing a rigid linker and one RALA unit presents an adequate transfection efficacy and low toxicity (based on MTT and apoptosis assays), however, the platform containing two RALA units and a flexible linker demonstrated high transfection efficacy while having modest toxicity in HER3 positive breast cancer cells. This may pave the way for further innovative applications of recombinant biopolymer when stable and economical productions need to be definitely considered. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [99mTc]Tc-ZHER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that ZHER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

HER2‐targeted therapy, especially the anti‐HER2 antibody trastuzumab, is standard for HER2‐positive breast cancer; however, its efficacy is limited in a subpopulation of patients. HER3 ligand (heregulin)‐dependent HER2‐HER3 interactions play a critical role in the evasion of apoptosis and are therefore a target for oncotherapy to treat HER2‐positive breast cancer. The anti‐HER2 antibody pertuzumab and anti‐HER3 antibody patritumab both target this heregulin–HER3‐HER2 complex in different ways. This study examined the anticancer efficacy of dual HER2 and HER3 blockade in trastuzumab‐resistant HER2‐positive breast cancer. HER2‐positive SKBR3 or BT474 cells overexpressing heregulin (SKBR3‐HRG, BT474‐HRG) were used to evaluate the efficacy of trastuzumab, pertuzumab, and patritumab in vitro by performing cell viability, immunoblotting, and clonogenic assays. The effects of these agents were then evaluated in vivo using BT474‐HRG and an intrinsic heregulin‐expressing and HER2‐positive JIMT‐1 xenograft models. SKBR3‐HRG and BT474‐HRG cells lost sensitivity to trastuzumab, which was accompanied by Akt activation. Unexpectedly, trastuzumab in combination with pertuzumab or patritumab also showed limited efficacy toward these cells. In contrast, trastuzumab/pertuzumab/patritumab triple treatment demonstrated potent anticancer efficacy, concomitant with strong repression of Akt. Finally, in heregulin‐expressing BT474‐HRG and JIMT‐1 xenograft models, the addition of pertuzumab and patritumab to trastuzumab also enhanced antitumor efficacy leading to tumor regression. The current study found that triple blockade of HER2 and HER3 using trastuzumab, pertuzumab, and patritumab could overcome resistance to trastuzumab therapy in heregulin‐expressing and HER2‐positive breast cancer, which could be exploited clinically. HER2‐targeted therapy, especially the anti‐HER2 antibody trastuzumab, is standard for HER2‐positive breast cancer; however, its efficacy is limited in a subpopulation of patients. The current study found that blockade of HER2 and HER3 using pertuzumab and patritumab could overcome resistance to trastuzumab therapy in heregulin‐expressing and HER2‐positive breast cancer, which could be exploited clinically. [ABSTRACT FROM AUTHOR]

Purpose To investigate the safety and clinical activity of comprehensive human epidermal growth factor receptor (HER) family receptor inhibition using lumretuzumab (anti-HER3) and pertuzumab (anti-HER2) in combination with paclitaxel in patients with metastatic breast cancer (MBC). Methods This phase Ib study enrolled 35 MBC patients (first line or higher) with HER3-positive and HER2-low (immunohistochemistry 1+ to 2+ and in-situ hybridization negative) tumors. Patients received lumretuzumab (1000 mg in Cohort 1; 500 mg in Cohorts 2 and 3) plus pertuzumab (840 mg loading dose [LD] followed by 420 mg in Cohorts 1 and 2; 420 mg without LD in Cohort 3) every 3 weeks, plus paclitaxel (80 mg/m2 weekly in all cohorts). Patients in Cohort 3 received prophylactic loperamide treatment. Results Diarrhea grade 3 was a dose-limiting toxicity of Cohort 1 defining the maximum tolerated dose of lumretuzumab when given in combination with pertuzumab and paclitaxel at 500 mg every three weeks. Grade 3 diarrhea decreased from 50% (Cohort 2) to 30.8% (Cohort 3) with prophylactic loperamide administration and omission of the pertuzumab LD, nonetheless, all patients still experienced diarrhea. In first-line MBC patients, the objective response rate in Cohorts 2 and 3 was 55% and 38.5%, respectively. No relationship between HER2 and HER3 expression or somatic mutations and clinical response was observed. Conclusions Combination treatment with lumretuzumab, pertuzumab and paclitaxel was associated with a high incidence of diarrhea. Despite the efforts to alter dosing, the therapeutic window remained too narrow to warrant further clinical development. Trial registration: on ClinicalTrials.gov with the identifier NCT01918254 first registered on 3rd July 2013. [ABSTRACT FROM AUTHOR]

Background: Cryobiopsy is recently being promoted for biopsy of tumors in the lung periphery in precision medicine for lung cancer; the obtained tissue samples have been reported to be more useful compared to those obtained using forceps, because of the larger volume and higher quality. However, the influence of freezing and thawing of tissues when performing cryobiopsy on the results of immunohistochemistry (IHC) has not been completely understood., Methods: In this study, consecutive patients who underwent diagnostic bronchoscopy with cryobiopsy for peripheral pulmonary lesions (PPLs) at our institution between June 2017 and November 2021 were reviewed retrospectively. Specimens of diagnosed cases of unresectable or recurrent non-small cell lung carcinoma (NSCLC) were selected. We compared the results of IHC assessment for programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2), and human epidermal growth factor receptor 3 (HER3) in cryobiopsy specimens versus conventional forceps biopsy specimens from the same site in the same procedure., Results: Twenty-four of 40 patients were male (60%). The most frequent histologic type of cancer was adenocarcinoma (n=31, 77.5%), followed by NSCLC (n=4, 10%), squamous cell carcinoma (n=3, 7.5%), and others (n=2, 5%). The concordance rates of the tumor proportion scores (TPSs) for PD-L1, IHC score for HER2 and, IHC scores for HER3 were 85%, 72.5%, and 75%, respectively; the weighted kappa were 0.835, 0.637, and 0.697, respectively., Conclusions: Freezing and thawing associated with cryobiopsy had virtually no effect on the results of IHC. We suggest that cryobiopsy specimens would therefore be ideal for precision medicine and translational research., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-621/coif). YO serves as an unpaid editorial board member of Translational Lung Cancer Research from January 2020 to December 2023. YM reports research fund from Hitachi and honoraria from Olympus, AstraZeneca, NOVARTIS, COOK, AMCO, Thermo Fisher Scientific, Erbe Elektromedizin GmbH, Fujifilm, Chugai, Eli Lilly, Merck and Takeda. TI reports grants from Hitachi High-Tech Corporation and honoraria from COOK, Chugai pharma, Eli Lilly, Olympus, Novartis pharma, Fujifilm and Thermo Fisher Scientific K.K. KU reports grants from Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Nos. JP22K15698 JP19K16966), and receives payments for lectures from Novartis, Thermo Fisher Scientific, AstraZeneca and Chugai. TY reports lecture fees from AstraZeneca, Chugai, Ono, Eli Lilly, and Takeda, and research funds from AMGEN, AstraZeneca, Takeda, Daiichi-Sankyo, Ono, MSD, AbbVie, Novartis, Chugai, Novartis, Chugai, Merck, Blueprint, and BMS. YG reports grants from AZK, Pfizer, Abbvie, Eli Lilly, Pfizer, Bristol Myers Squibb, Ono, Novartis, Kyorin, DaiichiSankyo, Novartis, and Prefered Networ, honoraria from Eli Lilly, Chugai,Taiho, Boehringer Ingelheim, Ono, Bristol Myers Squibb, Pfizer, MSD, Novartis. Merck, and Thermo Fischer, participation on an advisory board from AstraZeneca, Chugai, Boehringer Ingelheim, Eli Lilly, Taiho, Pfizer, Novartis, Guardant Health Inc., Illumina, DaiichiSankyo, Ono Pharmaceutical, Bristol Myers Squibb, and MSD, and leadership in Cancer Net Japan, JAMT. HH serves as a consultant to AstraZeneca, Eli Lilly, Chugai, Roche, Ono, BMS, and MSD, and receives lecture fees from AstraZeneca, MSD, Eli Lilly Ono, BMS, Chugai, Roche, Kyowa-Kirin, and Novartis, and research funds from MSD, AstraZeneca, BMS, Ono, Merck Biopharma, Daiichi-Sankyo, Janssen, Genomic Health, Chugai, Roche, and Novartis. Yuichiro Ohe receives lecture fees from AstraZeneca and Chugai, and research funds from AstraZeneca, Chugai, Eli Lilly, Ono, BMS, Kyorin, Dainippon-Sumitomo, Pfizer, Taiho, Novartis, Takeda, Kissei, Daiichi-Sankyo, Janssen, and LOXO. YY reports honoraria for lectures from MDS, Chugai-pharma, AstraZeneca, Novartis, Pfizer, Thermo Fisher Science, and Amgen. The other authors have no conflicts of interest to declare., (2023 Translational Lung Cancer Research. All rights reserved.)

Background: This open-label, multicenter, phase Ib study assessed the safety and preliminary activity of duligotuzumab, a dual-action antibody that blocks ligand binding to human epidermal growth factor receptor 3 (HER3) and epidermal growth factor receptor, in combination with chemotherapy, in the first-line treatment of patients with recurrent/metastatic squamous cell cancer of the head and neck.Methods: On day 1, duligotuzumab at a dose of 1650 mg intravenously was combined with cisplatin at a dose of 100 mg/m2 and 5-fluorouracil at a dose of 1000 mg/m2 /day on days 1 to 4 in treatment arm A, or carboplatin (area under the curve, 6 mg/mL/min) and paclitaxel (at a dose of 200 mg/m2 ) in treatment arm B. Up to 6 cycles (21 days/cycle) were followed by duligotuzumab maintenance until disease progression or intolerable toxicity occurred.Results: Nine patients in arm A and 15 patients in arm B received a median of 6 cycles of chemotherapy, and a median of 11 cycles (arm A) and 9 cycles (arm B) of duligotuzumab. Dose-limiting toxicities occurred in 3 patients in arm A and 1 patient in arm B. Grade ≥ 3 treatment-related adverse events (graded according to graded according to National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]) in ≥ 3 patients were neutropenia (5 patients), hypokalemia (4 patients), dehydration (3 patients), anemia (3 patients), and diarrhea (3 patients) in arm A, and neutropenia (8 patients), anemia (5 patients), febrile neutropenia (4 patients), leukopenia (3 patients), thrombocytopenia (3 patients), and hypomagnesemia (3 patients) in arm B. The chemotherapy dose was reduced in 19 of 24 patients. Sixteen patients (67%) demonstrated objective responses regardless of human papillomavirus status or neuregulin 1 (NRG1) mRNA expression (arm A: 2 confirmed complete responses and 4 confirmed partial responses; arm B: 2 confirmed complete responses and 8 confirmed partial responses).Conclusions: Duligotuzumab in combination with cisplatin/5-fluorouracil or carboplatin/paclitaxel demonstrated encouraging activity in patients with recurrent/metastatic squamous cell cancer of the head and neck; an association with increased frequency and severity of select adverse events relative to historical data was suggestive of the potentiation of chemotherapy-related adverse events. Cancer 2016;122:3803-3811. © 2016 American Cancer Society. [ABSTRACT FROM AUTHOR]

ABSTRACT Human epidermal growth factor receptor 3 (HER3) is a member of the human epidermal growth factor receptor (HER) family. The main characteristic of HER3 is that it does not possess tyrosine kinase activity, unlike other HERs. The role of HER3 in tumorigenesis has now been recognized, particularly in head and neck squamous cell carcinomas (HNSCCs). Despite conflicting studies, HER3 was found to be overexpressed in HNSCC samples, and correlates with disease progression and poor survival, especially when it is coexpressed with other HERs. HER3 is a significant factor in HNSCC treatment resistance. Indeed, HER3 is a major mechanism described for cetuximab resistance because of modification of epidermal growth factor receptor (EGFR) internalization and by phosphotidylinositol-3-kinase (PI3K)/AKT signaling pathway activation. HER3 also affects resistance to tyrosine kinase inhibitors (TKIs) and thereby promotes treatment escape and radiotherapy resistance by activation of the survival signaling pathway. To counteract this, pharmacologic inhibitors of HER3 are currently in development and could significantly improve HNSCC treatment. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2412-E2418, 2016 [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 3 (HER3) is a unique member of the human epidermal growth factor receptor (HER/EGFR) family, since it has negligible kinase activity. Therefore, HER3 must interact with a kinase-proficient receptor to form a heterodimer, leading to the activation of signaling cascades. Overexpression of HER3 is observed in various human cancers, including non-small cell lung cancer (NSCLC), and correlates with poor clinical outcomes in patients. Studies on the underlying mechanism demonstrate that HER3-initiated signaling promotes tumor metastasis and causes treatment failure in human cancers. Upregulation of HER3 is frequently observed in EGFR -mutant NSCLC treated with EGFR-tyrosine kinase inhibitors (TKIs). Increased expression of HER3 triggers the so-called EGFR-independent mechanism via interactions with other receptors to activate "bypass signaling pathways", thereby resulting in resistance to EGFR-TKIs. To date, no HER3-targeted therapy has been approved for cancer treatment. In both preclinical and clinical studies, targeting HER3 with a blocking antibody (Ab) is the only strategy being examined. Recent evaluations of an anti-HER3 Ab-drug conjugate (ADC) show promising results in patients with EGFR-TKI-resistant NSCLC. Herein, we summarize our understanding of the unique biology of HER3 in NSCLC refractory to EGFR-TKIs, with a focus on its dimerization partners and subsequent activation of signaling pathways. We also discuss the latest development of the therapeutic Abs and ADCs targeting HER3 to abrogate EGFR-TKI resistance in NSCLC., (© 2023 The Author(s).)

Background: Although heregulin and human epidermal growth factor receptor 3 (HER3) are frequently expressed at high levels in patients with head and neck cancer, their prognostic value remains unclear. The authors explored the prognostic significance of heregulin/HER3 expression in patients with oropharyngeal squamous cell carcinoma (OPSCC), taking into account other HER family members as well as p16 status.Methods: Ninety-six primary tumor specimens from patients with OPSCC were retrospectively collected and analyzed for heregulin messenger RNA (mRNA) using in situ hybridization and for HER3, epidermal growth factor receptor, and human epidermal growth factor receptor 2 (HER2) using quantitative immunohistochemistry. Heregulin and HER3 mRNA levels were also examined among different tumor types using The Cancer Genome Atlas database.Results: High heregulin mRNA (> the median) correlated significantly with poor overall survival (OS) (hazard ratio [HR], 8.48; 95% confidence interval [95% CI], 2.17-33.17 [P =.002]) but not disease-free survival (HR, 1.52; 95% CI, 0.64-3.65 [P =.341]) in patients with OPSCC. Heregulin mRNA correlated negatively with OS in both patients with p16-positive (P =.049) and p16-negative (P =.091) OPSCC on univariate analysis. High HER3 (> the median) also correlated with poor OS (HR, 4.68; 95% CI, 1.47-14.90 [P =.009]) on multivariate analysis. Epidermal growth factor receptor levels independently correlated with disease-free survival (P =.025) and inversely correlated with p16 status (P =.012). In addition, The Cancer Genome Atlas data demonstrated that head and neck squamous cell carcinoma exhibits higher heregulin expression compared with other solid tumor types examined.Conclusions: High heregulin mRNA and high HER3 protein levels were found to independently correlate with poor OS in patients with OPSCC. These data support targeting HER3 in patients with heregulin-high OPSCC and warrant further clinical investigation. [ABSTRACT FROM AUTHOR]

Specific inhibition of members of the EGFR (epidermal growth factor receptor) family, particularly EGFR and HER2 (human epidermal growth factor receptor 2), are an important therapeutic strategy in many human cancers. Compared with classical chemotherapy, these targeted therapeutics are very specific and initially effective, but acquired resistance against these targeted therapies is a recurring threat. A growing body of recent work has highlighted a pseudokinase in the EGFR family, HER3, and its ligand, NRG (neuregulin β1), to be of importance in models of resistant cancers, as well as in patients. In the present article, we describe some of the roles in which HER3 can mediate acquired resistance and discuss the current efforts to target HER3 itself in cancer. [ABSTRACT FROM AUTHOR]

Highlights: [•] Simultaneous engagement of functional arms in monoclonal and multispecific antibodies is essential component of their activity. [•] Optimization of cross-arm binding efficiency increases the potency of therapeutic antibodies. [•] Computational modeling prioritizes design approaches for increasing cross-arm binding. [ABSTRACT FROM AUTHOR]

Kinases catalyse the phosphorylation of target substrates on hydroxy group-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Through this biochemical activity, kinases play critical roles in many signal transduction and disease pathways. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive on the basis of mutations of key conserved active-site residues. However, recent biochemical and structural analyses have revealed several enzymatically active pseudokinases, suggesting either that novel mechanisms of phosphorylation are at play or that the constraints for highly conserved active-site residues are looser than originally anticipated. The purpose of the present review is to summarize several of the active pseudokinases, and one in particular termed KSR (kinase suppressor of Ras), which was recently found to possess a kinase activity that can become accelerated through an allosteric mechanism. Utilization of catalytic activity or structural features of the kinase fold may be key to the function of many pseudokinases. [ABSTRACT FROM AUTHOR]

HER2 (human epidermal growth factor receptor-2)-amplified tumours are characterized by constitutive signalling via the HER2-HER3 co-receptor complex. Although phosphorylation activity is driven entirely by the HER2 kinase, signal volume generated by the complex is under the control of HER3, and a large capacity to increase its signalling output accounts for the resiliency of the HER2-HER3 tumour driver and accounts for the limited efficacies of anti-cancer drugs designed to target it. In the present paper we describe deeper insights into the dynamic nature ofHER3 signalling. Signalling output by HER3 is under several modes of regulation, including transcriptional, posttranscriptional, translational, post-translational and localizational control. These redundant mechanisms can each increase HER3 signalling output and are engaged in various degrees depending on how the HER3/PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signalling network is disturbed. The highly dynamic nature of HER3 expression and signalling, and the plurality of downstream elements and redundant mechanisms that function to ensure HER3 signalling throughput identify HER3 as a major signalling hub in HER2-amplified cancers and a highly resourceful guardian of tumorigenic signalling in these tumours. [ABSTRACT FROM AUTHOR]

Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling. [Copyright &y& Elsevier]

Purpose: This study investigated the safety and clinical activity of lumretuzumab, a humanised antihuman epidermal growth factor receptor 3 (HER3) monoclonal antibody, in combination with carboplatin and paclitaxel in first-line treatment of patients with squamous non-small cell lung cancer (sqNSCLC). HER3 ligand heregulin and HER3 protein expression were evaluated as potential biomarkers of clinical activity., Patients and Methods: This open-label, phase Ib/II study enrolled patients receiving lumretuzumab at 800 mg (flat) in combination with carboplatin (area under the curve (AUC) 6 mg/mL×min) and paclitaxel (200 mg/m 2 ) administered intravenously on a every 3-week schedule. Adverse event (AE) rates and tumour responses were determined. Heregulin messenger RNA (mRNA) and HER3 protein expression were investigated in archival tumour biopsies., Results: Altogether, 12 patients received lumretuzumab in combination with carboplatin and paclitaxel. The most frequent AEs were gastrointestinal, haematological and nervous system toxicities, which were generally mild and manageable. Partial responses were observed in 3 of 12 patients lasting 81, 177 and 207 days. All responses were achieved in tumours expressing higher heregulin mRNA levels., Conclusion: Lumretuzumab in combination with carboplatin and paclitaxel was well tolerated. Objective responses were enriched in tumours expressing higher heregulin mRNA levels., Competing Interests: Competing interests: WJ, MC, MH and MW are the sponsor employees and have sponsor stock ownership. CA and FM are also the sponsor employees. IJ is the sponsor consultant from A4P. AC is the member of the Speaker Bureau of Roche and got research support from Roche.

The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGβ1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib., Competing Interests: Competing interestsAuthors declare no conflicts of interest or competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)