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Zhuang, et al.
agent could be removed after spheroidal construction, Another notable study on interrogating the
these paramagnetic agents, at high concentration, could functional dependencies and cellular interactions in the
be detrimental to cell survival. Comparatively, another brain tumor was conducted by Tang et al. They developed
label-free alternative using acoustic wave has also been a 3D-printed glioblastoma (GBM) model consisting
investigated. By applying non-destructive SAW, Chen of patient-derived glioblastoma stem cells (GSCs),
et al. assembled large amounts of cell spheroids in a astrocytes, and neural stem cells (NSCs), with or without
fluidic environment, forming various patterns within the presence of macrophages in a blended GelMA and
a couple of seconds [177] . By altering the acoustic wave glycidyl methacrylate-HA hydrogel through digital light
frequency, several geometric patterns ranging from processed-based bioprinting [180] . The printed construct
circle, square, and line to complex geometries could was composed of 2 regions: (i) GSC or mixed GSC/
be obtained. A complete fusion of fibroblast spheroids, macrophage encapsulated tumor cores, (ii) surrounded by a
HUVEC spheroids, and co-cultured spheroids were non-neoplastic region enriched with NSCs and astrocytes.
observed within 72h. To explore this versatility and The upregulation of the glioblastoma tissue-specific gene
modularity, the selected strategy was further applied sets, as compared to 2D and GSC spheres, suggested a
to generate densely packed hepatic tissue constructs better dynamic viewing window of transcriptional states
from fibroblast, HUVECs and primary rat hepatocytes. than ex vivo-derived glioblastoma tissues. Further, the
Notably, the formation of bile canaliculi was observed inclusion of macrophages resulted in upregulation of
at the hepatic junctions over a 6-day culture. The hypoxic response and glycolic metabolism, eliciting
results highlighted a remarkable effectiveness of this invasiveness signatures in the tetra-culture brain tumor
contactless, biocompatible, and label-free method in model. This significantly indicated that the 3D-printed
assembling spheroids through a highly efficient process. GBM model, to a higher extent, resembled the pathologic
Interestingly, Parfenov et al. recently reported a hybrid conditions in vivo.
magnetoacoustic bioassembly method that allowed The TME comprises numerous signaling molecules
rapid assembly of 3D tissue construction from spheroids and resulting pathways that influence the angiogenic
within a medium, with a relatively low concentration of response. Achieving a durable and efficient antiangiogenic
paramagnetic agent (gadolinium salt) [178] . Harnessing response will require approaches that simultaneously and/
the strength from both magnetic waves and acoustic or sequentially target multiple aspects of the TME [181] .
sounds, this method not only circumvents a challenge Dey et al. developed a 3D-vascularized breast cancer
from a potentially adverse effect stemming from the micro-environment that consists of HUVECs, metastatic
paramagnetic agent, but also imparts more flexibility on MDA-MB-231 cells, and fibroblasts-laden fibrin gel
the assembled structure. as the tumor stroma to investigate the interactions
The major challenge associated with replicating between cellular and acellular components in a TME [182] .
tumor models is simulating the heterogeneity of cellular Given the critical role of matrix stiffness in regulating
components and ECM. Dynamic interactions between cells tumorigenesis, matrix density affecting angiogenesis and
and the ECM contribute to tumor initiation, progression, invasion was investigated by varying the fibrinogen and
and metastasis through biophysiochemical cues. For thrombin concentration. The impact of fibroblasts was
instance, macrophages are a heterogeneous population examined by embedding the pre-vascularized spheroids
of cells that are crucial to the detection, phagocytosis, into a fibrin matrix, which was pre-loaded with fibroblasts
and destruction of pathogens. However, when recruited in a serial density from 0.25 million to 2 million cells/
to tumor cells, the TAMs polarize differently and do not mL. Interestingly, an increase in total vessel length and
display an anti-inflammatory role but rather facilitate branching index was observed with increasing fibroblast
tumor growth and angiogenesis. In understanding the densities ranging from 0.25 million to 1 million cells/
crosstalk between glioblastoma cells and macrophages, mL, indicating enhanced angiogenesis. Furthermore, to
Heinrich et al. generated a 3D-bioprinted mini-brain mimic a vascularized TME, HUVECs were introduced
with GelMA/gelatin encapsulating mouse glioblastoma into the fibrin matrix with fibroblasts in a 2:1 ratio to
cells (GL261) in the core of a mouse macrophage cell create a vascular bed for tumor cells. Over a 7-day
line (RAW264.7)-enriched at the peripheral [179] . Over culture period, HUVECs that sprouted from the laden
a 4-day culture, active migration of macrophages tumor spheroid anastomosed with the vascularized fibrin
toward tumor cells was observed, while tumors also matrix, organizing into a wide range of capillaries. Cancer
displayed migration behavior toward macrophages, cells were observed within capillary networks, indicating
albeit less dramatic, indicating that macrophages could their intravasation. Taken together, these multicellular
be actively recruited by tumor cells and polarized into a bioprinted tumor models, with exquisite control on both
Glioblastoma-associated macrophages (GAMs) specific cellular and acellular components, serving as promising
phenotype. platforms to interrogate cellular crosstalk, cell-to-

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