HER2-positive breast cancer accounts for approximately one-quarter of breast malignancies. This cancer type is driven by the amplification of the ERBB2 gene. Although targeted agents are used, resistance to treatment and varied therapeutic responses remain serious problems. Traditional research methods, such as two-dimensional cultures and xenografts, have provided molecular information. These older models cannot, however, reproduce the intricate structural and signaling organization found in human tumours. New experimental systems were developed to better represent human tumour physiology. These systems include organoids and breast cancer-on-chip devices. They are intended to provide more applicable avenues of research into HER2 signaling and drug responses.

HER2-positive organoids provide experimental systems that are considered more physiologically relevant than conventional cultures. These models, which include cell-line derived and patient-derived organoids, maintain features of the original tissue. These features include native polarity, interactions with the extracellular matrix, and intratumoral heterogeneity. This structure permits analysis of HER2 signaling dynamics. It also allows study of drug penetration within realistic gradients. Microfluidic breast cancer-on-chip approaches are used to integrate vascular and immune components under controlled flow. These systems support studies on antibody delivery, immune cell engagement, and adaptive resistance. By combining genetic fidelity with microenvironmental control, these methodologies are altering preclinical HER2 research.

Cell-line derived organoids are used to study how tumour architecture affects signaling. Recent analyses compared BT474 and SKBR3 organoids, as both are HER2-positive lines. Large differences in tissue organization and receptor signaling were found. BT474 cells were observed to form compact, spherical aggregates. These structures had defined polarity and reduced AKT phosphorylation. In contrast, SKBR3 cultures developed loosely organized structures. These showed increased HER2 expression but diminished cohesion. Confocal microscopy revealed that HER2 receptor density decreases toward the center of larger aggregates in both models. These morphological differences show how structural organization modulates HER2 activity. Tumour geometry and cell-to-cell contact appear to affect oncogenic signaling.

Drug screening studies show that antibody-based therapies behave differently in a three-dimensional architecture. The cytotoxic effect of a HER2-directed immunotoxin was tested. Its effect was found to be several orders of magnitude lower in HER2-positive cell-line derived organoids compared to 2D cultures. This difference was attributed to limited diffusion. The compound was restricted to the outer layers of the organoid, so central cells were largely unaffected. Similar findings were reported for trastuzumab. Microfluidic devices that generate linear drug gradients showed large differences in concentration-dependent responses. These observations suggest that reduced drug penetration may be a reason for resistance seen in clinical settings. This is particularly true in dense tumour regions that have poor vascularization.

A “tetraculture” model is a type of multicellular organoid. It is designed to incorporate stromal and immune components, which expands the predictive power of cell-line derived organoids. This model combines HER2-positive epithelial cells with fibroblasts, macrophages, and endothelial cells. The resulting structures were found to recapitulate patient-specific tissue organization. The localization of fibroblasts and macrophages differed depending on the tumour subtype. As an illustration, in HER2-positive BT474 organoids, cancer-associated fibroblasts aggregated in the center. This created compact structures that contained hypoxic cores. In contrast, SKBR3 organoids showed a more dispersed stromal pattern and peripheral macrophage accumulation. These different arrangements produced spatial gradients in viability and metabolic activity.

Patient-derived organoids (PDOs) are established directly from patient tissue, including metastatic biopsies. A main feature of these models is that they preserve the histological and molecular identity of the original tumour. This includes ERBB2 amplification, hormone receptor status, and other genomic alterations. PDOs provide an opportunity to observe patient-specific tumour behaviour under controlled experimental conditions. They also maintain the genetic diversity that defines clinical heterogeneity. Organoid biobanks have shown that these cultures retain the architecture, receptor expression, and growth characteristics of their tissue of origin. In addition, PDOs have been shown to maintain genomic stability across passages, which makes them suited for longitudinal analysis.

Patient-derived organoids are used for direct assessment of therapeutic sensitivity on tissues from a patient. This provides a route to individualized treatment design. One 2022 study used organoids to guide therapy in a metastatic case, achieving a complete clinical response. These models are also used to study resistance. Organoids grown from tumours that had lost HER2 after therapy were analyzed. These cultures required neuregulin-1 for growth, which revealed adaptive signaling changes. This provided a model to evaluate HER2-independent treatment strategies. PDOs also permit combination therapy evaluation. Studies using organoids from HER2-low and HER2-amplified tumours compared different drugs. Although trastuzumab alone often had limited cytotoxicity, adding pan-HER inhibitors like neratinib increased growth inhibition and delayed recovery.

A main advantage of microfluidic breast cancer-on-chip systems is the ability to reproduce vascular perfusion. This creates a situation that reflects drug transport and clearance. One study developed a chip where breast cancer–fibroblast organoids were enclosed by self-assembled human endothelial networks. Chemotherapeutic agents were delivered through these perfused microvessels. Under flow conditions, cytotoxic responses were observed to be markedly weaker and more gradual than in static cultures. This finding suggests reduced overall drug accumulation within the tumour tissue. Another study reported diminished cancer cell death under continuous perfusion, even with equivalent drug concentrations. These studies show how fluid dynamics affect therapeutic exposure. Conventional static assays may overestimate efficacy.

Microfluidic chips provide fine spatial control, which is used to study interactions between tumour cells and stromal components. One design used a chip with two adjacent chambers separated by microscale pillars. One side was seeded with MCF-7 cancer cells and the other with normal fibroblasts. This porous divider permitted cell-to-cell contact and molecular crosstalk. It was shown that co-culture with tumour cells induced fibroblast activation to a pro-tumorigenic myofibroblast state. This process resembles the desmoplastic reaction seen in actual tumours. Other chip systems are used to examine cancer cell migration and invasion in real time. A central region holds a spheroid of tumour cells embedded in matrix, while adjacent channels contain collagen or stromal cells. Investigators can monitor tumour cells invading out of the spheroid and quantify how different conditions affect this invasion.

Yes, both patient-derived organoids and chip systems are used for immunotherapy research. HER2-expressing PDOs have been co-cultured with chimeric antigen receptor (CAR) natural killer (NK) or T cells. In these experiments, the engineered immune cells successfully penetrated the organoid matrix. They also selectively eliminated HER2-positive cells. This method shows measurable antitumor efficacy and cytokine release patterns. Breast cancer-on-chip platforms are also used. Systems combining vascularized HER2-positive tumour organoids with a perfusable endothelial interface have been developed. This setup allows for real-time tracking of immune cell extravasation as cells traverse the endothelial layer. It is used to evaluate the effectiveness and selectivity of HER2-directed CAR-T and CAR-NK cells in a human-specific context.